Exogenous Hydrogen Peroxide Contributes to Heme Oxygenase-1 Delaying Programmed Cell Death in Isolated Aleurone Layers of Rice Subjected to Drought Stress in a cGMP-Dependent Manner.
ABSTRACT: Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that plays a dual role in plant cells. Here, we discovered that drought (20% polyethylene glycol-6000, PEG)-triggered decreases of HO-1 transcript expression and HO activity. However, exogenous H2O2 contributed toward the increase in HO-1 gene expression and activity of the enzyme under drought stress. Meanwhile, the HO-1 inducer hematin could mimic the effects of the H2O2 scavengers ascorbic acid (AsA) and dimethylthiourea (DMTU) and the H2O2 synthesis inhibitor diphenyleneiodonium (DPI) for scavenging or diminishing drought-induced endogenous H2O2. Conversely, the zinc protoporphyrin IX (ZnPPIX), an HO-1-specific inhibitor, reversed the effects of hematin. We further analyzed the endogenous H2O2 levels and HO-1 transcript expression levels of aleurone layers treated with AsA, DMTU, and DPI in the presence of exogenous H2O2 under drought stress, respectively. The results showed that in aleurone layers subjected to drought stress, when the endogenous H2O2 level was inhibited, the effect of exogenous H2O2 on the induction of HO-1 was enhanced. Furthermore, exogenous H2O2-activated HO-1 effectively enhanced amylase activity. Application of 8-bromoguanosine 3',5'-cyclic guanosine monophosphate (8-Br-cGMP) (the membrane permeable cGMP analog) promoted the effect of exogenous H2O2-delayed PCD of aleurone layers in response to drought stress. More importantly, HO-1 delayed the programmed cell death (PCD) of aleurone layers by cooperating with nitric oxide (NO), and the delayed effect of NO on PCD was achieved via mediation by cGMP under drought stress. In short, in rice aleurone layers, exogenous H2O2 (as a signaling molecule) triggered HO-1 and delayed PCD via cGMP which possibly induced amylase activity under drought stress. In contrast, as a toxic by-product of cellular metabolism, the drought-generated H2O2 promoted cell death.
Project description:Cereal aleurone layers undergo a gibberellin (GA)-regulated process of programmed cell death (PCD) following germination. Heme oxygenase-1 (HO-1) is known as a rate-liming enzyme in the degradation of heme to biliverdin IX?, carbon monoxide (CO), and free iron ions (Fe(2+)). It is a critical component in plant development and adaptation to environment stresses. Our previous studies confirmed that HO-1 inducer hematin (Ht) promotes the germination of rice seeds in drought (20% polyethylene glycol-6000, PEG) conditions, but the corresponding effects of HO-1 on the alleviation of germination-triggered PCD in GA-treated rice aleurone layers remain unknown. The present study has determined that GA co-treated with PEG results in lower HO-1 transcript levels and HO activity, which in turn results in the development of vacuoles in aleurone cells, followed by PCD. The pharmacology approach illustrated that up- or down-regulated HO-1 gene expression and HO activity delayed or accelerated GA-induced PCD. Furthermore, the application of the HO-1 inducer Ht and nitric oxide (NO) donor sodium nitroprusside (SNP) not only activated HO-1 gene expression, HO activity, and endogenous NO content, but also blocked GA-induced rapid vacuolation and accelerated aleurone layers PCD under drought stress. However, both HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) and NO scavenger 2-(4-carboxyphenyl0-4, 4,5,5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO) reserved the effects of Ht and SNP on rice aleurone layer PCD under drought stress by down-regulating endogenous HO-1 and NO, respectively. The inducible effects of Ht and SNP on HO-1 gene expression, HO activity, and NO content were blocked by cPTIO. Together, these results clearly suggest that HO-1 is involved in the alleviation of GA-induced PCD of drought-triggered rice aleurone layers by associating with NO.
Project description:H2S is a signaling molecule in plants and animals. Here we investigated the effects of H2S on programmed cell death (PCD) in barley (Hordeum vulgare L.) aleurone layers. The H2S donor NaHS significantly delayed PCD in aleurone layers isolated from imbibed embryoless barley grain. NaHS at 0.25?mM effectively reduced the accumulation of superoxide anion (·O2 (-)), hydrogen peroxide (H2O2), and malondialdehyde (MDA), promoted the activity of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and decreased those of lipoxygenase (LOX) in isolated aleurone layers. Quantitative-PCR showed that NaHS treatment of aleurone tissue led to enhanced transcript levels of the antioxidant genes HvSOD1, HvAPX, HvCAT1, and HvCAT2 and repressed transcript levels of HvLOX (lipoxygenase gene) and of two cysteine protease genes HvEPA and HvCP3-31. NaHS treatment in gibberellic acid- (GA-) treated aleurone layers also delayed the PCD process, reduced the content of ·O2 (-), and increased POD activity while decreasing LOX activity. Furthermore, ?-amylase secretion in barley aleurone layers was enhanced by NaHS treatment regardless of the presence or absence of GA. These data imply that H2S acted as an antioxidant in delaying PCD and enhances ?-amylase secretion regardless of the presence of GA in barley aleurone layers.
Project description:Haem oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, it is confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1 protein expression exhibited lower levels in GA-treated layers, whereas the hydrogen peroxide (H(2)O(2)) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H(2)O(2) either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with the HO-1 specific inhibitor, zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of the HO-1 inducer, haematin, and the enzymatic reaction product of HO, carbon monoxide (CO) aqueous solution, both of which brought about a noticeable induction of HO expression, substantially prevented GA-induced PCD. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by haematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in a decrease in H(2)O(2) production and a delay in PCD. In addition, the antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able not only to delay PCD but also to mimic the effects of haematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H(2)O(2) production.
Project description:BACKGROUND:Vacuolar processing enzymes (VPEs) have been identified as the enzymes that regulate vacuole-mediated programmed cell death (PCD) in plants. The mechanism that VPE regulates the PCD in rice aleurone layers remains unknown. RESULTS:The aleurone layers treated with distilled water exerted caspase-1 and VPE activity, both of which were inhibited by the caspase-1 specific inhibitor Ac-YVAD-CMK but not by the caspase-3 specific inhibitor Ac-DEVD-CHO. However, the caspase-1 and caspase-3 inhibitors weakened the activity of caspase-3. Combined with the effects of endogenous gibberellin (GA) on the induction of OsVPEs, we suggest that the OsVPE3 in the aleurone layers, which exhibits caspase-1-like activity, is a key molecule in GA-induced PCD via regulating the protease with caspase-3-like activity. Many studies have confirmed that vacuolar fusion is an important feature of vacuole-mediated PCD in plants. In this experiment, the process of vacuole fusion was accompanied by changes in the structure of actin filaments (AFs), specifically, their depolymerization and polymerization. The process of vacuolar fusion was accelerated or delayed by the promotion or inhibition of the depolymerization of AFs, respectively. Here, the inhibition of OsVPE3 blocked the depolymerization of AFs and delayed the fusion of vacuoles, indicating that OsVPE3 can regulate the fusion of vacuoles in rice aleurone layers via mediating AFs. Furthermore, the depolymerization of AFs contributed to the up-regulation of OsVPE3 gene expression and VPE activity, resulting in accelerated PCD in rice aleurone layers. However, the inhibitor of VPE reversed the effects of AF depolymerization on the activity of VPE, then postponing the process of PCD, implying that AF can involve in GA-induced PCD of rice aleurone layers by mediating OsVPE3. CONCLUSIONS:Together, activation of OsVPE3 and depolymerization of AFs shortened the process of vacuolation and PCD in rice aleurone layers, and OsVPE3 interacted with AFs during regulation.
Project description:Background and Aims:Although several studies have confirmed the beneficial roles of exogenous melatonin in lateral root (LR) formation, the molecular mechanism is still elusive. Here, the role of hydrogen peroxide (H2O2) in the induction of LR formation triggered by melatonin was investigated. Methods:Alfalfa (Medicago sativa 'Biaogan') and transgenic Arabidopsis seedlings were treated with or without melatonin, diphenyleneiodonium (DPI, NADPH oxidase inhibitor), N,N'-dimethylthiourea (DMTU, H2O2 scavenger), alone or combined. Then, H2O2 content was determined with 2',7'-dichlorofluorescein diacetate (H2DCFDA)-dependent fluorescence and spectrophotography. Transcript levels of cell cycle regulatory genes were analysed by real-time reverse transcription-PCR. Key Results:Application of exogenous melatonin not only increased endogenous H2O2 content but also induced LR formation in alfalfa seedlings. Consistently, melatonin-induced LR primordia exhibited an accelerated response. These inducible responses were significantly blocked when DPI or DMTU was applied. Compared with the wild-type, transgenic Arabidopsis plants overexpressing alfalfa MsSNAT (a melatonin synthesis gene) increased H2O2 accumulation and thereafter LR formation, both of which were blocked by DPI or DMTU. Similarly, melatonin-modulated expression of marker genes responsible for LR formation, including MsCDKB1;1, MsCDKB2;1, AtCDKB1;1 and AtCDKB2;1, was obviously impaired by the removal of H2O2 in both alfalfa and transgenic Arabidopsis plants. Conclusions:Pharmacological and genetic evidence revealed that endogenous melatonin-triggered LR formation was H2O2-dependent.
Project description:Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.
Project description:Drought-induced (Di19) proteins played important roles in plant growth, development, and abiotic stress responses. In the present study, a total of seven Di19 genes were identified in soybean. Each soybean Di19 gene showed specific responses to salt, drought, oxidative, and ABA stresses based on expression profiles. With a relatively higher transcript level among Di19 members under four stress treatments, GmDi19-5 was selected for detailed analysis. Inhibitor assays revealed that ABA inhibitor (Fluridone) or H2O2 inhibitor (DMTU) was involved in the drought- or salt-induced transcription of GmDi19-5. The GUS activity driven by the GmDi19-5 promoter was induced by salt, PEG, ABA, and MV treatments and tended to be accumulated in the vascular bundles and young leaves. A subcellular localization assay showed that GmDi19-5 protein localized in the nucleus. Further investigation showed that GmDi19-5 protein was involved in the interaction with GmLEA3.1. Overexpression of GmDi19-5 increased sensitivity of transgenic Arabidopsis plants to salt, drought, oxidative, and ABA stresses and regulated expression of several ABA/stress-associated genes. This present investigation showed that GmDi19-5 functioned as a negative factor under abiotic stresses and was involved in ABA and SOS signaling pathway by altering transcription of stress-associated genes.
Project description:Susceptibility of plants to abiotic stresses, including extreme temperatures, salinity and drought, poses an increasing threat to crop productivity worldwide. Here the drought-induced response of maize was modulated by applications of methyl jasmonate (MeJA) and salicylic acid (SA) to seeds prior to sowing and to leaves prior to stress treatment. Pot experiments were conducted to ascertain the effects of exogenous applications of these hormones on maize growth, physiology and biochemistry under drought stress and well-watered (control) conditions. Maize plants were subjected to single as well as combined pre-treatments of MeJA and SA. Drought stress severely affected maize morphology and reduced relative water content, above and below-ground biomass, rates of photosynthesis, and protein content. The prolonged water deficit also led to increased relative membrane permeability and oxidative stress induced by the production of malondialdehyde (from lipid peroxidation), lipoxygenase activity (LOX) and the production of H2O2. The single applications of MeJA and SA were not found to be effective in maize for drought tolerance while the combined pre-treatments with exogenous MeJA+SA mitigated the adverse effects of drought-induced oxidative stress, as reflected in lower levels of lipid peroxidation, LOX activity and H2O2. The same pre-treatment also maintained adequate water status of the plants under drought stress by increasing osmolytes including proline, total carbohydrate content and total soluble sugars. Furthermore, exogenous applications of MeJA+SA approximately doubled the activities of the antioxidant enzymes catalase, peroxidase and superoxide dismutase. Pre-treatment with MeJA alone gave the highest increase in drought-induced production of endogenous abscisic acid (ABA). Pre-treatment with MeJA+SA partially prevented drought-induced oxidative stress by modulating levels of osmolytes and endogenous ABA, as well as the activities of antioxidant enzymes. Taken together, the results show that seed and foliar pre-treatments with exogenous MeJA and/or SA can have positive effects on the responses of maize seedlings to drought.
Project description:Although studies have shown the concomitant occurrence of autophagic and programmed cell death (PCD) in plants, the relationship between autophagy and PCD and the factors determining this relationship remain unclear. In this study, seedlings of the wheat cultivar Jimai 22 were used to examine the occurrence of autophagy and PCD during polyethylene glycol (PEG)-8000-induced drought stress. Autophagy and PCD occurred sequentially, with autophagy at a relatively early stage and PCD at a much later stage. These findings suggest that the duration of drought stress determines the occurrence of PCD following autophagy. Furthermore, the addition of 3-methyladenine (3-MA, an autophagy inhibitor) and the knockdown of autophagy-related gene 6 (ATG6) accelerated PEG-8000-induced PCD, respectively, suggesting that inhibition of autophagy also results in PCD under drought stress. Overall, these findings confirm that wheat seedlings undergo autophagic survival under mild drought stress, with subsequent PCD only under severe drought.
Project description:BACKGROUNDS:The perturbance of chloroplast proteins is a major cause of photosynthesis inhibition under drought stress. The exogenous application of 5-aminolevulinic acid (ALA) mitigates the damage caused by drought stress, protecting plant growth and development, but the regulatory mechanism behind this process remains obscure. RESULTS:Wheat seedlings were drought treated, and the iTRAQ-based proteomic approach was employed to assess the difference in chloroplast protein content caused by exogenous ALA. A total of 9499 peptides, which could be classified into 2442 protein groups, were identified with ?0.01 FDR. Moreover, the contents of 87 chloroplast proteins was changed by drought stress alone compared to that of the drought-free control, while the contents of 469 was changed by exogenous ALA application under drought stress compared to that of drought stress alone. The Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results suggested that the ALA pretreatment adjusted some biological pathways, such as metabolic pathways and pathways involved in photosynthesis and ribosomes, to enhance the drought resistance of chloroplasts. Furthermore, the drought-promoted H2O2 accumulation and O2- production in chloroplasts were alleviated by the exogenous pretreatment of ALA, while peroxidase (POD) and glutathione peroxidase (GPX) activities were upregulated, which agreed with the chloroplast proteomic data. We suggested that ALA promoted reactive oxygen species (ROS) scavenging in chloroplasts by regulating enzymatic processes. CONCLUSIONS:Our results from chloroplast proteomics extend the understanding of the mechanisms employed by exogenous ALA to defend against drought stress in wheat.