Rice Cell Suspension Research Articles

Effects of Hydrogen Sulfide on Alternative Pathway Respiration and Induction of Alternative Oxidase Gene Expression in Rice Suspension Cells

The toxic effects of H2S on plants are well documented. However, the molecular mechanisms reponsible for inhibition of plants by H2S are still not completely understood. We determined the effects of NaHS in the range of 0.5-10 mM on the growth of rice suspension culture cells, as well as on the expression of the alternative oxidase (AOX) gene. AOX is the terminal oxidase of the alternative pathway (AP) and exists in plant mitochondria. The results showed that H2S treatment enhanced the AP activity. During the process of H2S treatment for 4 h, the AP activity increased dramatically and achieved the peak value at a concentration of 2 mM NaHS. Then it declined at higher concentrations of NaHS (5-10 mM) and maintained a steady level. The AOX1 gene transcript level also showed a similar change as the AP activity. Interestingly, different NaHS concentrations seemed to have different effects on the expression of AOX1a, AOX1b, and AOX1c. The induction of AOX expression by low concentrations of NaHS was inferred through a reactive oxygen species (ROS)-independent pathway. At the same time, rice cells grown in culture were very sensitive to H2S, different H2S concentrations induced an increase in the cell viability. These results indicate that the H2S-induced AOX induction might play a role in inhibiting the ROS production and have an influence on cell viability.

Differential metabolic response of cultured rice (Oryza sativa) cells exposed to high‐ and low‐temperature stress

Global mean temperatures are expected to rise by 2-4.5 degrees C by 2100, accompanied by an increase in frequency and amplitude of extreme temperature events. Greater climatic extremes and an expanded range of cultivation will expose rice to increasing stress in the future. Understanding gene expression in disparate thermal regimes is important for the engineering of cultivars with tolerance to nonoptimal temperatures. Our study investigated the proteomic responses of rice cell suspension cultures to sudden temperature changes. Cell cultures grown at 28 degrees C were subjected to 3-day exposure to 12 or 20 degrees C for low-temperature stress, and 36 or 44 degrees C for high-temperature stress. Quantitative label-free shotgun proteomic analysis was performed on biological triplicates of each treatment. Over 1900 proteins were expressed in one or more temperature treatments, and, of these, more than 850 were found to be responsive to either of the temperature extremes. These temperature-responsive proteins included more than 300 proteins which were uniquely expressed at either 12 or 44 degrees C. Our study also identified 40 novel stress-response proteins and observed that switching between the classical and the alternative pathways of sucrose metabolism occurs in response to extremes of temperature.

N-linked glycan analysis of glycoproteins secreted from rice cell suspension cultures under sugar starvation

Recently, the rice α-amylase 3D promoter system activated under sucrose-starved conditions has emerged as an alternative system for the production of useful recombinant proteins. However, the influence of N-linked glycan structures related to sugar starvation conditions has not yet been investigated. In this study, we determined the N-linked glycans of glycoproteins and human granulocyte-macrophage colony stimulating factor secreted from rice cell suspension cultures under sugar starvation conditions. The N-glycan patterns obtained from glycoproteins grown in growth medium with sucrose (+S) were different from those obtained under sucrose starvation (−S). The mass spectra of +S glycans exhibited eleven N-glycan patterns. Among these, the most abundant glycans were GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc (27.4% of total), followed by Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, GlcNAcβ1-2Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, and Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, each of which represented 15–17% of the total. Except for Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, all +S N-glycans were complex-types containing β1,3-galactose or α1,4-fucose. The five N-glycans that disappeared under −S conditions were GlcNAcβ1-2Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, Galβ1-3GlcNAcβ1-2Manα1-6(Galβ1-3GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAc, and Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc. The N-glycan pattern of glycoproteins included hGM-CSF derived from rice suspension cell culture, and all N-glycans contained plant-specific β1,2-xylose, core α1,3-fucose, and some Lewis a epitopes. High-mannose type N-glycans were not detected under either condition (−S or +S) among the secreted glycoproteins.

ChemInform Abstract: Synthesis and Biological Activity of 3′-Chloro, -Bromo, and -Iodoabscisic Acids, and Biological Activity of 3′-Fluoro-8′-hydroxyabscisic Acid.

Abstract (+)-3′-Chloro, -bromo and -iodoabscisic acids were synthesized, and their biological activities and metabolism were examined, as well as that of (+)-3′-fluoroabscisic acid. This was in order to estimate the effects of altered electron density at the C-2′ position of abscisic acid on both biological activity and metabolic stability. The biological activities of (+)-3′-chloro and -bromoabscisic acids were similar to, or higher than, those of (+)-abscisic acid in four bioassays, while (+)-3′-iodoabscisic acid was more potent in two bioassays and less potent than (+)-abscisic acid in the other two bioassays. The biological activities of 3′-haloabscisic acids seem to be dependent on the 3′-halogens, but not on the electron density at C-2′. The metabolism of (+)-3′-haloabscisic acids to ca. 50% of their original levels in the media of rice cell suspension culture was similar to that of (+)-abscisic acid. Metabolites from (+)-3′-chloro, -bromo and -iodoabscisic acids were not found in the culture media and cells, while (+)-3′-fluoroabscisic acid gave (+)-3′-fluoro-8′-hydroxyabscisic acid as a stable metabolite. (+)-3′-fluoro-8′-hydroxyabscisic acid showed biological activities lower than (+)-3′-fluoroabscisic acid, but higher than (−)-3′α-Fluorophaseic acid. These findings suggested that the biological activity of (+)-8′-hydroxyabscisic acid is intermediate between those of (+)-abscisic acid and (−)-phaseic acid. Abscisic acid may be inactivated in a stepwise manner by metabolism in plants.

Metabolome Analysis of Response to Oxidative Stress in Rice Suspension Cells Overexpressing Cell Death Suppressor Bax Inhibitor-1

Bax inhibitor-1 (BI-1) is a cell death suppression factor widely conserved in higher plants and animals. Overexpression of Arabidopsis BI-1 (AtBI-1) in plants confers tolerance to various cell death-inducible stresses. However, apart from the cell death-suppressing activity, little is known about the physiological roles of BI-1-overexpressing plants. In this study, we evaluated the effects of AtBI-1 overexpression on the rice metabolome in response to oxidative stress. AtBI-1-overexpressing rice cells in suspension displayed enhanced tolerance to menadione-induced oxidative stress compared with vector control cells, whereas AtBI-1 overexpression did not influence the increase of intracellular H(2)O(2) concentration or inhibition of oxidative stress-sensitive aconitase activity. Capillary electrophoresis-mass spectrometry (CE-MS)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed rice cells, e.g. depletion of the central metabolic pathway, imbalance of the redox state and energy charge, and accumulation of amino acids. Furthermore, comparative metabolome analysis demonstrated that AtBI-1 overexpression did not affect primary metabolism in rice cells under normal growth conditions but significantly altered metabolite composition within several distinct pathways under cell death-inducible oxidative stress. The AtBI-1-mediated metabolic alteration included recovery of the redox state and energy charge, which are known as important factors for metabolic defense against oxidative stress. These observations suggest that although AtBI-1 does not affect rice metabolism directly, its cell death suppression activity leads to enhanced capacity to acclimate oxidative stress.

Application of commercial cryoprotectants for the cryopreservation of transgenic rice suspension cells

Application of commercial cryoprotectants for the cryopreservation of transgenic rice suspension cells

Molecular characterization of OsPAP2: transgenic expression of a purple acid phosphatase up-regulated in phosphate-deprived rice suspension cells

A phosphate starvation-induced, purple, acid phosphatase cDNA was cloned from rice, Oryza sativa. The cDNA encoding the phosphatase (OsPAP2) has 1,893 bp with an open reading frame of 630 amino acid residues. The deduced amino acid sequence of OsPAP2 shows identities of 60-63% with other plant purple acid phosphatases and appears to have five conserved motifs containing the residues involved in metal binding. OsPAP2 expression is up-regulated in the rice plant and in cell cultures in the absence of phosphate (P( i )). The induced expression of OsPAP2 is a specific response to P( i ) starvation, and is not affected by the deprivation of other nutrients. OsPAP2 expression was responsive to the level of P( i )-supply, and transcripts of OsPAP2 were abundant in P( i )-deprived roots. The OsPAP2 cDNA was expressed as a 69 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsPAP2 gene was introduced into Arabidopsis via an Agrobacterium-mediated transformation. Functional expression of the OsPAP2 gene in the transgenic Arabidopsis line was confirmed by northern and western blot analyses, as well as by phosphatase activity assays. These results suggest that the OsPAP2 gene can be used to develop new transgenic dicotyledonous plants that are able to adapt to P( i )-deficient conditions.

Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l −1 at day 10.

Enhanced delivery of siRNA complexes by sonoporation in transgenic rice cell suspension cultures.

Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic- T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEI) at a mass ratio of 1:10 (0.33 microg siRNA:3.3 microg PEI) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419 W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

The calmodulin-binding transcription factor OsCBT suppresses defense responses to pathogens in rice.

We previously isolated the OsCBT gene, which encodes a calmodulin (CaM)-binding protein, from a rice expression library constructed from fungal elicitor-treated rice suspension cells. In order to understand the function of OsCBT in rice, we isolated and characterized a T-DNA insertion mutant allele named oscbt-1. The oscbt-1 mutant exhibits reduced levels of OsCBT transcripts and no significant morphological changes compared to wild-type plant although the growth of the mutant is stunted. However, oscbt-1 mutants showed significant resistance to two major rice pathogens. The growth of the rice blast fungus Magnaporthe grisea, as well as the bacterial pathogen Xanthomonas oryzae pv. oryzae was significantly suppressed in oscbt-1 plants. Histochemical analysis indicated that the hypersensitive-response was induced in the oscbt-1 mutant in response to compatible strains of fungal pathogens. OsCBT expression was induced upon challenge with fungal elicitor. We also observed significant increase in the level of pathogenesis-related genes in the oscbt-1 mutant even under pathogen-free condition. Taken together, the results support an idea that OsCBT might act as a negative regulator on plant defense.

Rice isoflavone reductase‐like protein is associated with root development and ROS stress resistance

Isoflavone phytoalexins and isoflavone reductase gene in the rice has not been identified and characterized. We characterized OsIRL protein using Northern, Western, 2‐DE, immunohistochemical analysis, and transgenic plant in rice. The transcript and proteins were more abundant in stems and roots than in flower and leaves. Expression of OsIRL was more accumulated in the root than in the shoot and seed. To investigate the release of reactive oxygen intermediates (ROI) by seed during germination, we utilized the apoplastic oxidation of 2¡‐,7¡‐dichlorofluorescin to fluorescent 2¡‐7¡‐dichloroflurescein and DAB staining as an in vivo assay. Expression of OsIRL was increased in the root elongation and maturation position carrying accumulation of ROI. Expression of OsIRL was increased in the oxidants (H2O2, FeCl3, MV) and decreased in the antioxidants such as reduced glutathione in the root and rice suspension cell. OsIRL was accumulated in the root sclerenchyma, outside of cortex, endodermis, and metaphloem cell. A certain concentration of H2O2 was necessary for root elongation, suggesting that OsIRL protein may play a role as antioxidant. The OsIRL transcripts levels were highly increased in callus, leaves, stem, and roots by treatment with ABA. The transgenic OsIRL plants have methyl viologen resistance, ROS‐generator, indicating that OsIRL have involved in root growth and resistance to oxidative stress.

Auxin regulation in the interaction between Rice stripe virus and rice

AbstractThe expression of the YUCAA1 gene and the amount of endogenous indole acetic acid (IAA) in rice (Oryza sativa subsp. japonica) plants and rice suspension cells infected by Rice stripe virus (RSV) were investigated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and high-performance liquid chromatography, respectively. The results showed that the expression of the YUCAA1 gene and the amount of endogenous IAA increased at various times (16, 32, 48 and 64 h) after infection of rice suspension cells by RSV. In rice plants infected with RSV, the expression of the YUCAA1 gene and the amount of endogenous IAA increased, in comparison with healthy rice plants, at 4–8 days after infection, and decreased at 12 and 16 days. These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally, the expression of the RSV gene CP increased 2.9 times in rice plants after they were treated with a KPSC buffer to deplete the endogenous auxins, and decreased 45% after treatment with 30 μmol/l IAA. All of these results suggest that auxin may play a role in RSV replication in rice plants.

Subcutaneous Four-Week Repeated Dose Toxicity Studies of Rice Cell-Derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor in Rats.

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 µg/kg subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.

Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells

We have developed a method by which remarkably higher efficiencies of transient and stable transformation were achieved in bombardment transformation of plants. Over fivefold increase in transient gus gene expression was achieved when rice or maize suspension cells were bombarded with gold particles coated with plasmid DNA in the presence of protamine instead of the conventional spermidine. A 3.3-fold improvement in stable transformation efficiency was also observed using rice suspension cells with the new coating approach. The coated protamine-plasmid DNA complex resisted degradation by a DNase or by rice cell extract much longer than the spermidine-plasmid DNA complex. The results from this study suggest that protamine protects plasmid DNA longer than spermidine when being delivered inside the cells, probably by forming a nano-scale complex, and thus helps improve the efficiency of particle bombardment-mediated plant transformation.

Effects of inoculation methods and flask size on production of hctla4ig in transgenic rice cell suspension cultures

Effects of inoculation methods and flask size on production of hctla4ig in transgenic rice cell suspension cultures

Bioreactor operation under anoxia for enhanced production of hCTLA4Ig in 5-L stirred tank reactor transgenic rice cell suspension cultures

Bioreactor operation under anoxia for enhanced production of hCTLA4Ig in 5-L stirred tank reactor transgenic rice cell suspension cultures

Increased production of hctla4ig using in situ product recovery in transgenic rice cell suspension cultures

Increased production of hctla4ig using in situ product recovery in transgenic rice cell suspension cultures

Shear protective effects of Pluronic F-68 for production of hCTLA4Ig in transgenic rice cell suspension cultures

Shear protective effects of Pluronic F-68 for production of hCTLA4Ig in transgenic rice cell suspension cultures

Effects of proline on cell viability and hCTLA4Ig production in transgenic rice cell suspension cultures

Effects of proline on cell viability and hCTLA4Ig production in transgenic rice cell suspension cultures

Effects of substitute carbon sources on cell viability and hCTLA4Ig productivity in transgenic rice cell suspension cultures

Effects of substitute carbon sources on cell viability and hCTLA4Ig productivity in transgenic rice cell suspension cultures