Eiji Okuma

Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells

Environmental stress, including heavy metal stress, can cause oxidative damage to plants. Up-regulation of the antioxidant defense system induced by proline and glycinebetaine (betaine) alleviates the damaging effects of oxidative stress in plants. Here, we investigated the protective effects of exogenously applied proline and betaine on growth, accumulation of proline and betaine, lipid peroxidation and activity of antioxidant enzymes in cultured tobacco Bright Yellow-2 (BY-2) cells exposed to cadmium (Cd) stress. Cadmium stress (at 100 microM Cd) caused a significant inhibition of the growth of BY-2 cells, and both proline and betaine significantly mitigated this inhibition. In addition, the mitigating effect of proline was more pronounced than that of betaine. Cadmium stress leads to an accumulation of Cd and endogenous proline in cultured cells, increased lipid peroxidation and peroxidase (POX) activity, and decreased activity of superoxide dismutase (SOD) and catalase (CAT). Exogenous application of proline resulted in a decrease in lipid peroxidation and an increase in SOD and CAT activities without reducing Cd contents under Cd stress, while application of betaine resulted in a decrease in lipid peroxidation and an increase in CAT activity with reducing Cd accumulation. Furthermore, exogenous proline and betaine intensified the accumulation of proline and betaine in Cd-stressed BY-2 cells, respectively. The present study suggests that proline and betaine confer tolerance to Cd stress in tobacco BY-2 cells by different mechanisms.

Effects of exogenous application of proline and betaine on the growth of tobacco cultured cells under saline conditions

Abstract The effects of the application of exogenous proline and betaine on the growth of tobacco cultured cells subjected to salt stress were investigated. Both proline and betaine mitigated the inhibition of growth of tobacco cells under saline conditions, but the harmful effect of salinity was less reduced by betaine than by proline. The amount of intracellular betaine in tobacco cells cultured in the NaCI medium supplemented with 20 mm betaine was larger than that of intracellular proline in the NaCI medium with 20 mm proline. The 1,1-diphenyl2-picrylhydrazyl (DPPH) experiments showed that proline displayed an antioxidant activity and that the antioxidant activity of betaine was not detectable. The malondialdehyde (MDA) assay demonstrated that exogenous proline but not betaine decreased the amount of MDA in salt-stressed tobacco cells. These results suggest that the difference in the mitigation effects between proline and betaine may be responsible for the difference in the antioxidant activity.

Cooperative Function of PLDδ and PLDα1 in Abscisic Acid-Induced Stomatal Closure in Arabidopsis

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K+ channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

Exogenous proline mitigates the inhibition of growth of Nicotiana tabacum cultured cells under saline conditions

Abstract The addition of exogenous proline (10 mm) to Na100-saline culture medium, modified LS medium (Linsmaier and Skoog 1965: Physiol. Plant., 18, 100–127) with 100 mm NaCl promoted the growth of tobacco (Nicotiana tabacum L., cv. Bright Yellow-2) suspension cells unadapted to salt stress without maintaining a high ratio of K+ to Na+ ions under salinity conditions. The addition of exogenous glutamic acid or alanine were not comparable to that of exogenous proline. The proline contents of the NaCl-unadapted cells became much higher when the cells were grown in Na100-saline culture medium with 10 mm proline than when the cells were cultured without proline. The accumulation of K+, Na+, counter ions was sufficient to compensate for the increase of the water potential of the cells caused by salinity. These results suggest that exogenous proline does not act as a nitrogen store and that proline may act as a protectant for enzymes and membranes against salt inactivation rather than as a compatible solute in tobacco suspension cells.

bHLH Transcription Factors That Facilitate K+ Uptake During Stomatal Opening Are Repressed by Abscisic Acid Through Phosphorylation

Decreasing the transcription of genes encoding K+ channels contributes to inhibition of stomatal opening in Arabidopsis. Aperture Control Light induces the opening of stomata, pores in leaf epidermis that enable gas and water vapor exchange, whereas the hormone abscisic acid (ABA) causes stimulation of stomatal closure and inhibition of stomatal opening. Takahashi et al. identified a family of transcription factors called ABA-responsive kinase substrates (AKSs) that transcribed genes encoding potassium channels that promoted stomatal opening. ABA induced the phosphorylation of AKS transcription factors, preventing them from binding to the promoters of K+ channel–encoding genes and inducing a decrease in stomatal opening. Thus, AKS-mediated transcription stimulates stomatal opening, a process that is antagonized by ABA-induced phosphorylation of AKS transcription factors. Stomata open in response to light and close after exposure to abscisic acid (ABA). They regulate gas exchange between plants and the atmosphere, enabling plants to adapt to changing environmental conditions. ABA binding to receptors initiates a signaling cascade that involves protein phosphorylation. We show that ABA induced the phosphorylation of three basic helix-loop-helix (bHLH) transcription factors, called AKSs (ABA-responsive kinase substrates; AKS1, AKS2, and AKS3), in Arabidopsis guard cells. In their unphosphorylated state, AKSs facilitated stomatal opening through the transcription of genes encoding inwardly rectifying K+ channels. aks1aks2-1 double mutant plants showed decreases in light-induced stomatal opening, K+ accumulation in response to light, activity of inwardly rectifying K+ channels, and transcription of genes encoding major inwardly rectifying K+ channels without affecting ABA-mediated stomatal closure. Overexpression of potassium channel in Arabidopsis thaliana 1 (KAT1), which encodes a major inwardly rectifying K+ channel in guard cells, rescued the phenotype of aks1aks2-1 plants. AKS1 bound directly to the promoter of KAT1, an interaction that was attenuated after ABA-induced phosphorylation. The ABA agonist pyrabactin induced phosphorylation of AKSs. Our results demonstrate that the AKS family of bHLH transcription factors facilitates stomatal opening through the transcription of genes encoding inwardly rectifying K+ channels and that ABA suppresses the activity of these channels by triggering the phosphorylation of AKS family transcription factors.

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis.

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca(2+)-permeable channel currents by ABA or oscillation of the cytosolic free Ca(2+) concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca(2+) oscillation in ABA signal pathway of Arabidopsis guard cells.

Chitosan-induced stomatal closure accompanied by peroxidase-mediated reactive oxygen species production in Arabidopsis.

Chitosan induced stomatal closure in wild type-plants and NADPH oxidase knock-out mutants (atrbohD atrbohF), and reactive oxygen species (ROS) production in wild-type guard cells. Closure and production were completely abolished by catalase and a peroxidase inhibitor. These results indicate that chitosan induces ROS production mediated by peroxidase, resulting in stomatal closure.

OsHKT1;4-mediated Na+ transport in stems contributes to Na+ exclusion from leaf blades of rice at the reproductive growth stage upon salt stress

BackgroundNa+ exclusion from leaf blades is one of the key mechanisms for glycophytes to cope with salinity stress. Certain class I transporters of the high-affinity K+ transporter (HKT) family have been demonstrated to mediate leaf blade-Na+ exclusion upon salinity stress via Na+-selective transport. Multiple HKT1 transporters are known to function in rice (Oryza sativa). However, the ion transport function of OsHKT1;4 and its contribution to the Na+ exclusion mechanism in rice remain to be elucidated.ResultsHere, we report results of the functional characterization of the OsHKT1;4 transporter in rice. OsHKT1;4 mediated robust Na+ transport in Saccharomyces cerevisiae and Xenopus laevis oocytes. Electrophysiological experiments demonstrated that OsHKT1;4 shows strong Na+ selectivity among cations tested, including Li+, Na+, K+, Rb+, Cs+, and NH4+, in oocytes. A chimeric protein, EGFP-OsHKT1;4, was found to be functional in oocytes and targeted to the plasma membrane of rice protoplasts. The level of OsHKT1;4 transcripts was prominent in leaf sheaths throughout the growth stages. Unexpectedly however, we demonstrate here accumulation of OsHKT1;4 transcripts in the stem including internode II and peduncle in the reproductive growth stage. Moreover, phenotypic analysis of OsHKT1;4 RNAi plants in the vegetative growth stage revealed no profound influence on the growth and ion accumulation in comparison with WT plants upon salinity stress. However, imposition of salinity stress on the RNAi plants in the reproductive growth stage caused significant Na+ overaccumulation in aerial organs, in particular, leaf blades and sheaths. In addition, 22Na+ tracer experiments using peduncles of RNAi and WT plants suggested xylem Na+ unloading by OsHKT1;4.ConclusionsTaken together, our results indicate a newly recognized function of OsHKT1;4 in Na+ exclusion in stems together with leaf sheaths, thus excluding Na+ from leaf blades of a japonica rice cultivar in the reproductive growth stage, but the contribution is low when the plants are in the vegetative growth stage.

Regulation of a Proteinaceous Elicitor-induced Ca2+ Influx and Production of Phytoalexins by a Putative Voltage-gated Cation Channel, OsTPC1, in Cultured Rice Cells

Background: Molecular mechanisms for elicitor-induced changes in cytosolic Ca2+ concentration and its molecular link with regulation of phytoalexin biosynthesis in plant immunity remain mostly unknown. Results: TvX-induced Ca2+ influx and the phytoalexin accumulations were suppressed in Ostpc1 knock-out cells. Conclusion: OsTPC1 plays a role in TvX-induced Ca2+ influx consequently required for the regulation of phytoalexin biosynthesis. Significance: Voltage-dependent plasma membrane Ca2+-permeable channel activity of the plant TPC1 was shown for the first time. Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca2+ followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca2+, mainly due to a Ca2+ influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca2+-sensitive photoprotein, aequorin, TvX-induced Ca2+ elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca2+-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca2+ influx as a plasma membrane Ca2+-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.

Proline and Glycinebetaine Confer Cadmium Tolerance on Tobacco Bright Yellow-2 Cells by Increasing Ascorbate-Glutathione Cycle Enzyme Activities

Cadmium (Cd) stress significantly decreased membrane integrity and impaired the ascorbate (ASC)-glutathione (GSH) cycle in tobacco Bright Yellow-2 cells. Exogenous application of proline and glycinebetaine (betaine) significantly restored the membrane integrity and increased the activities of ASC-GSH cycle enzymes under Cd stress without maintenance of the rich ASC or GSH pools. Moreover, proline offered more efficient protection against Cd stress than betaine.