Wheat Protoplasts Research Articles

TaMYB3, Encoding a Functional MYB Transcriptor, Isolated from the Purple Pericarp of Triticum aestivum

Purple pericarp is an interesting and useful trait in Triticum aestivum, but the molecular mechanism behind this phenotype remains unclear. The allelic variation in the MYB transcriptors is associated with the phenotype of pigmented organs in many plants. In this study, a MYB transcription factor gene, TaMYB3, was isolated using homology-based cloning and a differentially expressed gene mining approach, to verify the function of the MYB transcriptor in the purple pericarp. The coding sequence of TaMYB3 in cultivar Gy115 was the same as that in cultivar Opata. TaMYB3 was localized to FL0.62–0.95 on chromosome 4BL. The TaMYB3 protein contains DNA-binding and transcription-activation domains, and clustered on a phylogenetic tree with the MYB proteins that regulates anthocyanin and proanthocyanin biosynthesis. TaMYB3 localized in the nuclei of Arabidopsis thaliana and wheat protoplasts after it was transiently expressed with PEG transformation. TaMYB3 induced anthocyanin synthesis in the pericarp cells of Opata in the dark in collaboration with the basic helix–loop–helix protein ZmR, which is also the function of ZmC1. However, TaMYB3 alone did not induce anthocyanin biosynthesis in the pericarp cells of the white grain wheat cultivar Opata in the light after bombardment, whereas the single protein ZmR did. Light increased the expression of TaMYB3 in the pericarp of Gy115 and Opata, but only induced anthocyanin biosynthesis in the grains of Gy115. Our results extend our understanding of the molecular mechanism of the purple pericarp trait in T. aestivum.

Molecular and Functional Characterization of Wheat ARGOS Genes Influencing Plant Growth and Stress Tolerance.

Auxin Regulated Gene involved in Organ Size (ARGOS) is significantly and positively associated with organ size and is involved in abiotic stress responses in plants. However, no studies on wheat ARGOS genes have been reported to date. In the present study, three TaARGOS homoeologous genes were isolated and located on chromosomes 4A, 4B, and 4D of bread wheat, all of which are highly conserved in wheat and its wild relatives. Comparisons of gene expression in different tissues demonstrated that the TaARGOSs were mainly expressed in the stem. Furthermore, the TaARGOS transcripts were significantly induced by drought, salinity, and various phytohormones. Transient expression of the TaARGOS-D protein in wheat protoplasts showed that TaARGOS-D localized to the endoplasmic reticulum. Moreover, overexpression of TaARGOS-D in Arabidopsis resulted in an enhanced germination rate, larger rosette diameter, increased rosette leaf area, and higher silique number than in wild-type (WT) plants. The roles of TaARGOS-D in the control of plant growth were further studied via RNA-seq, and it was found that 105 genes were differentially expressed; most of these genes were involved in ‘developmental processes.’ Interestingly, we also found that overexpression of TaARGOS-D in Arabidopsis improved drought and salinity tolerance and insensitivity to ABA relative to that in WT plants. Taken together, these results demonstrate that the TaARGOSs are involved in seed germination, seedling growth, and abiotic stress tolerance.

TaARPC3, Contributes to Wheat Resistance against the Stripe Rust Fungus.

The actin cytoskeleton participates in numerous cellular processes, including less-characterized processes, such as nuclear organization, chromatin remodeling, transcription, and signal transduction. As a key regulator of actin cytoskeletal dynamics, the actin related protein 2/3 complex (Arp2/3 complex) controls multiple developmental processes in a variety of tissues and cell types. To date, the role of the Arp2/3 complex in plant disease resistance signaling is largely unknown. Herein, we identified and characterized wheat ARPC3, TaARPC3, which encodes the C3 subunit of the Arp2/3 complex. Expression of TaARPC3 in the arc18 mutant of Saccharomyces cerevisiae Δarc18 resulted in complementation of stress-induced phenotypes in S. cerevisiae, as well as restore wild-type cell shape malformations. TaARPC3 was found predominantly to be localized in the nucleus and cytoplasm when expressed transiently in wheat protoplast. TaARPC3 was significantly induced in response to avirulent race of Puccinia striiformis f. sp. tritici (Pst). Knock-down of TaARPC3 by virus-induced gene silencing resulted in a reduction of resistance against Pst through a specific reduction in actin cytoskeletal organization. Interestingly, this reduction was found to coincide with a block in reactive oxygen species (ROS) accumulation, the hypersensitive response (HR), an increase in TaCAT1 mRNA accumulation, and the growth of Pst. Taken together, these findings suggest that TaARPC3 is a key subunit of the Arp2/3 complex which is required for wheat resistance against Pst, a process that is associated with the regulation of the actin cytoskeleton.

An UDP-Glucosyltransferase Gene from Barley Confers Disease Resistance to Fusarium Head Blight

UDP-glucosyltransferases (UGTs) contribute to Fusarium head blight (FHB) resistance of wheat and barley by glycosylating the deoxynivalenol (DON), which is produced by Fusarium fungus. In this study, seven alleles of barley HvUGT14077 (GenBank No.GU170356.1) were cloned using RT-PCR. Among them, HvUGT-10W1, which was isolated from a FHB resistant barley variety 10W1, was significantly up-regulated in young spikes after F. graminearum (F.g) inoculation. HvUGT-10W1::GFP was subcellularly located in the plasma membrane and cytoplasm of the wheat protoplasts. In vitro antifungal activity assay showed that the HvUGT-10W1 protein exerted obvious inhibition against the growth of F.g. The silencing of the HvUGT-10W1 by virus-induced gene silencing (VIGS) resulted in compromised FHB resistance of 10W1, which was shown by the increased infected colonies on the leaves. These indicated that the barley HvUGT-10W1 may also contribute to F.g resistance in barley and provided a potential candidate gene to develop transgenic barley with enhanced FHB resistance.

Bioinformatic Analyses of Subgroup-A Members of the Wheat bZIP Transcription Factor Family and Functional Identification of TabZIP174 Involved in Drought Stress Response.

Extensive studies in Arabidopsis and rice have demonstrated that Subgroup-A members of the bZIP transcription factor family play important roles in plant responses to multiple abiotic stresses. Although common wheat (Triticum aestivum) is one of the most widely cultivated and consumed food crops in the world, there are limited investigations into Subgroup A of the bZIP family in wheat. In this study, we performed bioinformatic analyses of the 41 Subgroup-A members of the wheat bZIP family. Phylogenetic and conserved motif analyses showed that most of the Subgroup-A bZIP proteins involved in abiotic stress responses of wheat, Arabidopsis, and rice clustered in Clade A1 of the phylogenetic tree, and shared a majority of conserved motifs, suggesting the potential importance of Clade-A1 members in abiotic stress responses. Gene structure analysis showed that TabZIP genes with close phylogenetic relationships tended to possess similar exon–intron compositions, and the positions of introns in the hinge regions of the bZIP domains were highly conserved, whereas introns in the leucine zipper regions were at variable positions. Additionally, eleven groups of homologs and two groups of tandem paralogs were also identified in Subgroup A of the wheat bZIP family. Expression profiling analysis indicated that most Subgroup-A TabZIP genes were responsive to abscisic acid and various abiotic stress treatments. TabZIP27, TabZIP74, TabZIP138, and TabZIP174 proteins were localized in the nucleus of wheat protoplasts, whereas TabZIP9-GFP fusion protein was simultaneously present in the nucleus, cytoplasm, and cell membrane. Transgenic Arabidopsis overexpressing TabZIP174 displayed increased seed germination rates and primary root lengths under drought treatments. Overexpression of TabZIP174 in transgenic Arabidopsis conferred enhanced drought tolerance, and transgenic plants exhibited lower water loss rates, higher survival rates, higher proline, soluble sugar, and leaf chlorophyll contents, as well as more stable osmotic potential under drought conditions. Additionally, overexpression of TabZIP174 increased the expression of stress-responsive genes (RD29A, RD29B, RAB18, DREB2A, COR15A, and COR47). The improved drought resistance might be attributed to the increased osmotic adjustment capacity. Our results indicate that TabZIP174 may participate in regulating plant response to drought stress and holds great potential for genetic improvement of abiotic stress tolerance in crops.

The wheat calcium-dependent protein kinase TaCPK7-D positively regulates host resistance to sharp eyespot disease.

Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, limits wheat production worldwide. Here, TaCPK7-D, encoding a subgroup III member of the calcium-dependent protein kinase (CPK) family, was identified from the sharp eyespot-resistant wheat line CI12633 through comparative transcriptomic analysis. Subsequently, the defence role of TaCPK7-D against R. cerealis infection was studied by the generation and characterization of TaCPK7-D-silenced and TaCPK7-D-overexpressing wheat plants. Rhizoctonia cerealis inoculation induced a higher transcriptional level of TaCPK7-D in the resistant wheat line CI12633 than in the susceptible cultivar Wenmai 6. The expression of TaCPK7-D was significantly induced after exogenous application of 1-aminocyclopropane-1-carboxylic acid (an ethylene biosynthesis precursor). The green fluorescent protein signal distribution assays indicated that TaCPK7-D localizes to the plasma membrane in both onion epidermal cells and wheat protoplasts. Following R. cerealis inoculation, TaCPK7-D-silenced wheat CI12633 plants displayed more severe sharp eyespot symptoms than control CI12633 plants. Four defence-associated genes (β-1,3-glucanase, chitinase 1, defensin and TaPIE1) and an ethylene biosynthesis key gene, ACO2, were significantly suppressed in the TaCPK7-D-silenced wheat plants compared with control plants. Conversely, TaCPK7-D-overexpressing wheat lines showed increased resistance to sharp eyespot compared with untransformed recipient wheat Yangmai 16. Furthermore, the transcriptional levels of these four defence-related genes and ACO2 gene were significantly elevated in TaCPK7-D-overexpressing plants compared with untransformed recipient wheat plants. These results suggest that TaCPK7-D positively regulates the wheat resistance response to R. cerealis infection through the modulation of the expression of these defence-associated genes, and that TaCPK7-D is a candidate to improve sharp eyespot resistance in wheat.

Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation

Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants.

Optimization Conditions of Wheat Mesophyll Protoplast Isolation

The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the FDA staining respectively. Results showed that, with the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%; with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h; considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h; meanwhile, with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min (replicated three times). So 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition.

TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis.

BackgroundNAC (NAM, ATAF, and CUC) transcription factors play important roles in plant biological processes, including phytohormone homeostasis, plant development, and in responses to various environmental stresses.MethodsTaNAC29 was introduced into Arabidopsis using the Agrobacterium tumefaciens-mediated floral dipping method. TaNAC29-overexpression plants were subjected to salt and drought stresses for examining gene functions. To investigate tolerant mechanisms involved in the salt and drought responses, expression of related marker genes analyses were conducted, and related physiological indices were also measured. Expressions of genes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsA novel NAC transcription factor gene, designated TaNAC29, was isolated from bread wheat (Triticum aestivum). Sequence alignment suggested that TaNAC29 might be located on chromosome 2BS. TaNAC29 was localized to the nucleus in wheat protoplasts, and proved to have transcriptional activation activities in yeast. TaNAC29 was expressed at a higher level in the leaves, and expression levels were much higher in senescent leaves, indicating that TaNAC29 might be involved in the senescence process. TaNAC29 transcripts were increased following treatments with salt, PEG6000, H2O2, and abscisic acid (ABA). To examine TaNAC29 function, transgenic Arabidopsis plants overexpressing TaNAC29 were generated. Germination and root length assays of transgenic plants demonstrated that TaNAC29 overexpression plants had enhanced tolerances to high salinity and dehydration, and exhibited an ABA-hypersensitive response. When grown in the greenhouse, TaNAC29-overexpression plants showed the same tolerance response to salt and drought stresses at both the vegetative and reproductive period, and had delayed bolting and flowering in the reproductive period. Moreover, TaNAC29 overexpression plants accumulated lesser malondialdehyde (MDA), H2O2, while had higher superoxide dismutase (SOD) and catalase (CAT) activities under high salinity and/or dehydration stress.ConclusionsOur results demonstrate that TaNAC29 plays important roles in the senescence process and response to salt and drought stresses. ABA signal pathway and antioxidant enzyme systems are involved in TaNAC29-mediated stress tolerance mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0644-9) contains supplementary material, which is available to authorized users.

Protein can be taken up by damaged wheat roots and transported to the stem

Proteins of animal origin can represent a portion of the overall nitrogen (N) pool in the soil environment and there is a possibility that plants may utilize animal proteins as a N source. Using wheat (Triticum aestivum L.) we investigated if the model protein, ovalbumin was taken up into the roots and transported within the plant. In roots, ovalbumin was associated with the epidermis when no root damage was evident, but with minor root damage, it was present in intercellular spaces throughout the cortex and at the endodermis. Ovalbumin was only found in the stem when minor damage to the root system was evident. Suspension cultures of wheat protoplasts revealed that ovalbumin was not assimilated into individual plant cells. Our results suggest that ovalbumin uptake and subsequent movement in wheat is possible only after root damage has occurred. Apoplastic movement may enable animal protein to enter plant tissues above the soil level where they could be consumed by grazers.

Association of VPg and eIF4E in the host tropism at the cellular level of Barley yellow mosaic virus and Wheat yellow mosaic virus in the genus Bymovirus

Barley yellow mosaic virus (BaYMV) and Wheat yellow mosaic virus (WYMV) are separate species in the genus Bymovirus with bipartite plus-sense RNA genomes. In fields, BaYMV infects only barley and WYMV infects only wheat. Here, we studied the replicative capability of the two viruses in barley and wheat mesophyll protoplasts. BaYMV replicated in both barley and wheat protoplasts, but WYMV replicated only in wheat protoplasts. The expression of wheat translation initiation factor 4E (eIF4E), a common host factor for potyviruses, from the WYMV genome enabled WYMV replication in barley protoplasts. Replacing the BaYMV VPg gene with that of WYMV abolished BaYMV replication in barley protoplasts, whereas the additional expression of wheat eIF4E from BaYMV genome restored the replication of the BaYMV mutant in barley protoplasts. These results indicate that both VPg and the host eIF4E are involved in the host tropism of BaYMV and WYMV at the replication level.

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies.

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV.

Fertile introgression products generated via somatic hybridization between wheat and Thinopyrum intermedium

Fertile hybrids were produced with genetic material transferred from Th. intermedium into a wheat background and supply a source of genetic variation to wheat improvement. Both symmetric and asymmetric somatic hybrids have been obtained from the combination of wheatgrass (Thinopyrum intermedium) and bread wheat (Triticum aestivum). Two wheat protoplast populations, one derived from embryogenic calli and the other from a non-regenerable, rapidly dividing cell line, were fused with Th. intermedium protoplasts which had been (or not been) pre-irradiated with UV. Among the 124 regenerated calli, 64 could be categorized as being of hybrid origin on the basis of plant morphology, peroxidase isozyme, RAPD DNA profiling and karyological analysis. Numerous green plantlets were regenerated from 13 calli recovered from either the symmetric hybrid (no UV pre-treatment) or the asymmetric one (30 s UV irradiation). One of these hybrid plants proved to be vigorous and self-fertile. The regenerants were all closer in phenotype to wheat than to Th. intermedium. Genomic in situ hybridization analysis showed that the chromosomes in the hybrids were largely intact wheat ones, although a few Th. intermedium chromosome fragments had been incorporated within them.

Construction of Whole Genome Radiation Hybrid Panels and Map of Chromosome 5A of Wheat Using Asymmetric Somatic Hybridization

To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.

Anoxia-induced elevation of cytosolic Ca2+ concentration depends on different Ca2+ sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.

High frequency of HMW-GS sequence variation through somatic hybridization between Agropyron elongatum and common wheat

A symmetric somatic hybridization was performed to combine the protoplasts of tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum). Fertile regenerants were obtained which were morphologically similar to tall wheatgrass, but which contained some introgression segments from wheat. An SDS-PAGE analysis showed that a number of non-parental high-molecular weight glutenin subunits (HMW-GS) were present in the symmetric somatic hybridization derivatives. These sequences were amplified, cloned and sequenced, to deliver 14 distinct HMW-GS coding sequences, eight of which were of the y-type (Hy1-Hy8) and six x-type (Hx1-Hx6). Five of the cloned HMW-GS sequences were successfully expressed in E. coli. The analysis of their deduced peptide sequences showed that they all possessed the typical HMW-GS primary structure. Sequence alignments indicated that Hx5 and Hy1 were probably derived from the tall wheatgrass genes Aex5 and Aey6, while Hy2, Hy3, Hx1 and Hy6 may have resulted from slippage in the replication of a related biparental gene. We found that both symmetric and asymmetric somatic hybridization could promote the emergence of novel alleles. We discussed the origination of allelic variation of HMW-GS genes in somatic hybridization, which might be the result from the response to genomic shock triggered by the merger and interaction of biparent genomes.

Apoplastic superoxide level in wheat protoplast under photooxidative stress is regulated by chloroplast redox signals: Effects on the antioxidant system

Apoplastic superoxide radical level in wheat mesophyll protoplasts was evaluated under different light intensities. Conditions that enhanced chloroplastic reactive oxygen species (ROS) production, such as high light intensity, paraquat treatments, or choloplastic Mn-superoxide dismutase (Mn-SOD) overexpression, increased the level of apoplastic superoxide. By contrast, the addition of diphenyl iodonium, a suicide NADPH oxidase inhibitor; DCMU, an herbicide that blocks plastoquinone reduction; and EGTA, a Ca 2+ chelator, decreased the superoxide level in the apoplast. Intracellular ROS production was rapidly stimulated by high light intensity and paraquat, and decreased in the presence of DCMU. The association between apoplastic superoxide level and total activities of the antioxidant enzymes superoxide dismutase (SOD) (E.C.:1.15.1.1), glutathione reductase (GR) (E.C.:1.6.4.2), ascobate peroxidase (APX) (E.C.:1.11.1.11), and catalase (CAT) (E.C.:1.11.1.6) was also evaluated. Changes in apoplastic superoxide level were positively correlated with activities of antioxidant enzymes mainly located in the chloroplasts, but not with CAT activity, which is located in peroxisomes and mitochondria. Regulation of apoplastic superoxide level by chloroplastic ROS and plastoquinone redox state is discussed, as well as its role in modulating antioxidant defense responses under photooxidative stress.

Cell Membrane Surface Potential (ψ 0) Plays a Dominant Role in the Phytotoxicity of Copper and Arsenate

Negative charges at cell membrane surfaces (CMS) create a surface electrical potential (psi(0)) that affects ion concentrations at the CMS and consequently affects the phytotoxicity of metallic cations and metalloid anions in different ways. The zeta potentials of root protoplasts of wheat (Triticum aestivum), as affected by the ionic environment of the solution, were measured and compared with the values of psi(0) calculated with a Gouy-Chapman-Stern model. The mechanisms for the effects of cations (H(+), Ca(2+), Mg(2+), Na(+), and K(+)) on the acute toxicity of Cu(2+) and As(V) to wheat were studied in terms of psi(0). The order of effectiveness of the ions in reducing the negativity of psi(0) was H(+) > Ca(2+) approximately Mg(2+) > Na(+) approximately K(+). The calculated values of psi(0) were proportional to the measured zeta potentials (r(2) = 0.93). Increasing Ca(2+) or Mg(2+) activities in bulk-phase media resulted in decreased CMS activities of Cu(2+) ({Cu(2+)}(0)) and increased CMS activities of As(V) ({As(V)}(0)). The 48-h EA50{Cu(2+)}(b) ({Cu(2+)} in bulk-phase media accounting for 50% inhibition of root elongation over 48 h) increased initially and then declined, whereas the 48-h EA50{As(V)}(b) decreased linearly. However, the intrinsic toxicity of Cu(2+) (toxicity expressed in terms of {Cu(2+)}(0)) appeared to be enhanced as psi(0) became less negative and the intrinsic toxicity of As(V) appeared to be reduced. The psi(0) effects, rather than site-specific competitions among ions at the CMS (invoked by the biotic ligand model), may play the dominant role in the phytotoxicities of Cu(2+) and As(V) to wheat.

Superoxide dismutase and glutathione reductase overexpression in wheat protoplast: photooxidative stress tolerance and changes in cellular redox state

In previous works, we have established a correlation between antioxidant system response and tolerance to drought, osmotic stress and photooxidative stress of different wheat cultivars with contrasting drought tolerance. In the present work, a protocol to obtain and transform wheat protoplasts was established. Transgenic protoplasts with Manganese Superoxide Dismutase (Mn-SOD) (E.C.: 1.15.1.1) and Glutathione Reductase (GR) (E.C.: 1.6.4.2) overexpression in chloroplasts were obtained, and their responses to photooxidative stress were characterized. Protoplasts with Mn-SOD or GR overexpression, showed different responses and tolerance to photooxidative stress. Protoplasts with Mn-SOD overexpression showed lower levels of oxidative damage, higher level of endogenous hydrogen peroxide and a great induction of total SOD and GR activities during photooxidative treatments. In protoplasts with GR overexpression the oxidative damage provoked by the photooxidative treatment was similar to control protoplasts, the GSH content and GSH/GSH + GSSG ratio were higher than control and Mn-SOD transformed protoplast, and total SOD and GR activities were not induced. Our results suggest that the differential responses and tolerance to photooxidative stress given by Mn-SOD or GR overexpression, also depend on the effects of these enzyme activities over the cellular redox state balance, which modulate the responses to photooxidative stress.

Topography and functional information of plasma membrane

By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8+/-0.09 nm in diameter and 1.9+/-0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40-60 nm, and a range of 1.8-5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12-40 nm for their diameter and 0.7-2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 microm(2). According to their size, we classified the invaginated pits into two types--larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead protoplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still detectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in diameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6-9.8 nm, thicker in regions covered with proteins.