Glucose-6-phosphate dehydrogenase and abscisic acid mediate programmed cell death induced by aluminum toxicity in soybean root tips.

Programmed cell death (PCD) induced by aluminum (Al) is considered an important reason of Al phytotoxicity. However, the underlying mechanism of how Al induces PCD remains largely unknown in plants. The roles of glucose-6-phosphate dehydrogenase (G6PDH) and abscisic acid (ABA) in regulating Al-induced PCD were investigated in soybean roots. Al treatment increased G6PDH activity, while inhibition of G6PDH activity alleviated PCD occurrence and reactive oxygen species (ROS) accumulation under Al stress. Overexpression of cytosolic G6PDH1 enhanced G6PDH activity, thus promoting ROS production and cell death under Al exposure. Inhibition of NADPH oxidase activity mitigated ROS generation and cell death under Al stress. Further investigation demonstrated that G6PDH positively regulated the activity of NADPH oxidase under Al treatment using pharmacological and transgenic approach. Furthermore, Al stress increased ABA production, while inhibition of ABA biosynthesis alleviated PCD occurrence and ROS accumulation under Al stress. Interestingly, ABA upregulated G6PDH1 expression and G6PDH activity under Al stress. These results suggest that G6PDH mediates Al-induced PCD occurrence through the activation of NADPH oxidase-dependent ROS production, and ABA acts upstream of G6PDH in this process. This study will provide novel clues for the improvement of Al phytotoxicity in acidic soils.

An Arabidopsis Nucleoporin NUP85 modulates plant responses to ABA and salt stress.

Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.

ahg12 is a dominant proteasome mutant that affects multiple regulatory systems for germination of Arabidopsis.

The ubiquitin-proteasome system is fundamentally involved in myriad biological phenomena of eukaryotes. In plants, this regulated protein degradation system has a pivotal role in the cellular response mechanisms for both internal and external stimuli, such as plant hormones and environmental stresses. Information about substrate selection by the ubiquitination machinery has accumulated, but there is very little information about selectivity for substrates at the proteasome. Here, we report characterization of a novel abscisic acid (ABA)-hypersensitive mutant named ABA hypersensitive germination12 (ahg12) in Arabidopsis. The ahg12 mutant showed a unique pleiotropic phenotype, including hypersensitivity to ABA and ethylene, and hyposensitivity to light. Map-based cloning identified the ahg12 mutation to cause an amino acid conversion in the L23 loop of RPT5a, which is predicted to form the pore structure of the 19S RP complex of the proteasome. Transient expression assays demonstrated that some plant-specific signaling components accumulated at higher levels in the ahg12 mutant. These results suggest that the ahg12 mutation led to changes in the substrate preference of the 26S proteasome. The discovery of the ahg12 mutation thus will contribute to elucidate the characteristics of the regulated protein degradation system.

Investigation of neurotoxic and immunotoxic effects of some plant growth regulators at subacute and subchronic applications on rats.

The present study was aimed to investigate the effects of subacute and subchronic treatment of some plant growth regulators (PGRs), such as abscisic acid (ABA) and gibberellic acid (GA3), on neurological and immunological biomarkers in various tissues of rats. The activities of acetylcholinesterase (AChE) and butrylcholinesterase (BChE) were selected as biomarkers for neurotoxic biomarkers. Adenosine deaminase (ADA) and myeloperoxidase (MPO) were measured as indicators for immunotoxic investigation purpose. Wistar albino rats were orally administered with 25 and 50 ppm of PGRs ad libitum for 25-50 days continuously with drinking water. The treatment of PGRs caused different effects on the activities of enzymes. Results showed that the administrations of ABA and GA3 increased AChE and BChE activities in some tissues of rats treated with both the dosages and periods of ABA and GA3. With regard to the immunotoxic effects, ADA activity fluctuated, while MPO activity increased after subacute and subchronic exposure of treated rat tissues to both dosages when compared with the controls. The observations presented led us to conclude that the administrations of PGRs at subacute and subchronic exposure increased AChE, BChE, and MPO activities, while fluctuating the ADA activity in various tissues of rats. This may reflect the potential role of these parameters as useful biomarkers for toxicity of PGRs.

Characterization and expression analysis of three CBF/DREB1 transcriptional factor genes from mangrove Avicennia marina.

Three CBF/DREB1 (C-repeat binding factor/dehydration responsive element-binding factor 1) homologues were isolated from mangrove Avicennia marina and designated AmCBF1, 2 and 3. Multiple sequence analysis showed that the three deduced proteins all contain an AP2 DNA-binding domain and two CBF signature sequences. According to the phylogenetic analysis, these proteins belong to the A-1 subgroup of the DREB subfamily. Expression analyses based on quantitative real-time PCR revealed that the AmCBF2 displayed relatively high expression under normal conditions, with the highest level in stems, while both AmCBF1 and 3 were weakly expressed without stress. The three genes also showed different responses to various environmental stimuli. The AmCBF2 was inducible by cold, drought, high salinity, heavy metals, as well as abscisic acid (ABA), and exhibited much stronger induction by cold, drought, Pb(2+) or Zn(2+) than by NaCl, ABA or Cd(2+). In contrast, both AmCBF1 and AmCBF3 displayed insignificant changes under these stimuli. These results indicate that the three AmCBF genes play different roles in A. marina and the AmCBF2 might be involved in the signaling pathway of cold, drought and heavy metal stress response.

Genome-wide identification and transcription analysis of soybean carotenoid oxygenase genes during abiotic stress treatments.

Carotenoid oxygenase is a key enzyme in carotenoid metabolism leading to the synthesis of two phytohormones, abscisic acid (ABA) and strigolactone, as well as norisoprenoids. Few studies have analyzed inter-relationship of the metabolic networks of these three substances. In this present paper, soybean carotenoid oxygenase genes were identified to reveal their phylogenetic relationships, and the transcriptional response of these genes to four abiotic stresses (NaCl, PEG, high and low temperature) and ABA treatment were investigated to characterize their potential roles in plant resistance. Positive selection was found in the branches of carotenoid cleavage dioxygenase (CCD1), CCD8 and NCED (9-cis-epoxycarotenoid oxygenase), indicating an adaptive evolution in these clades. In soybean eight carotenoid oxygenase genes were identified. The transcriptional responses of almost all of them under stress and ABA conditions were significantly altered when assessed by quantitative polymerase chain reaction. Notably, CCD1 and CCD4, previously known as the key genes in norisoprenoids metabolism, showed especially strong responses to the abiotic stresses and ABA treatment. Furthermore, transcription levels of CCD7 and CCD8, key genes for the strigolactone pathway, highly increased during ABA treatment providing further evidence that ABA is involved in regulating strigolactone metabolism. All of the carotenoid oxygenase genes in soybean are involved in plant abiotic stress physiology, and ABA is presumed to be a core regulatory substance. These findings provide some insights into the mechanisms that underlie the regulation of tolerance response to abiotic stresses in soybean.

Engineering the ABA plant stress pathway for regulation of induced proximity.

Chemically induced proximity (CIP) systems use small molecules and engineered proteins to control and study biological processes. However, small molecule-based systems for controlling protein abundance or activities have been limited by toxicity, instability, cost, and slow clearance of the small molecules in vivo. To address these problems, we modified proteins of the plant abscisic acid (ABA) stress response pathway to control the proximity of cellular proteins and showed that the system could be used to regulate transcription, signal transduction, and subcellular localization of proteins in response to exogenously applied ABA. We also showed that the ABA CIP system can be combined with other CIP systems to simultaneously control multiple processes. We found that, when given to mice, ABA was orally available and had a 4-hour half-life. These properties, along with its lack of toxicity and low cost, suggest that ABA may be well suited for therapeutic applications and as an experimental tool to control diverse cellular activities in vivo.

Evalution of toxicity of abcisic acid and gibberellic acid in rats: 50 days drinking water study.

In the present study, the influence of subchronic effects of two plant growth regulators (PGRs) [Abcisic acid (ABA) and Gibberellic acid (GA3)] on antioxidant defense systems [reduced glutathione (GSH), glutathione reductase (GR), superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT)] and lipid peroxidation level (malondialdehyde = MDA) in various tissues of the rat were investigated during treatment as a drinking water model. 75 ppm of ABA and GA3 in drinking water were continuously administered orally to rats (Sprague-Dawley albino) ad libitum for 50 days. The PGRs treatments caused different effects on the antioxidant defense systems and MDA content of dosed rats compared to controls. The lipid peroxidation end product MDA significantly increased in the lungs, heart and kidney of rats treated with GA3 without significant change in the spleen. ABA caused also a significant increase in MDA content in the spleen, lungs, heart and kidney. The GSH levels were significantly depleted in the spleen, lungs and stomach of rats treated with ABA without any change in the tissues of rats treated with GA3 except the kidney where it increased. Antioxidant enzyme activities such as SOD significantly increased in the lungs and stomach and decreased in the spleen and heart tissues of rats treated with GA3. Meanwhile, SOD significantly decreased in the spleen, heart and kidney and increased in the lungs of rats treated with ABA. While CAT activity significantly decreased in the lungs of rats treated with GA3, a significant increase occurred in the heart of rats treated with both PGRs. On the other hand, the ancillary enzyme GR activity in the tissues were either significantly depleted or not changed with PGRs treatment. The drug metabolizing enzyme GST activity significantly decreased in the lungs of rats treated with ABA but increased in the stomach of rats treated with both PGRs. As a conclusion, the rats resisted oxidative stress via the antioxidant mechanism. But the antioxidant mechanism could not prevent the increases in lipid peroxidation in rat's tissues. This data, along with changes, suggests that PGRs produced substantial systemic organ toxicity in the spleen, lungs, stomach, heart and kidney during a 50-day period of subchronic exposure.

Abcisic acid and gibberellic acid cause increased lipid peroxidation and fluctuated antioxidant defense systems of various tissues in rats.

The study was aimed at demonstrating changes in the antioxidant defense systems [Reduced glutathione (GSH), glutathione reductase (GR), superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT)] forming an antioxidative barrier and oxidative stress parameter (Malondialdehyde=MDA) in the various tissues of Sprague-Dawley rats which were administrated plant growth regulators (PGRs) [Abcisic acid (ABA) and Gibberellic acid (GA(3))] during 25 days. Seventy five parts per million of ABA and GA(3) as drinking water were administered orally to rats (Sprague-Dawley albino) ad libitum for 25 days continuously. The PGRs treatments caused different effects on antioxidant defense systems and MDA content of experimented rats compared to controls. The lipid peroxidation end product MDA significantly increased in the spleen and lungs of rats treated with ABA and GA(3) without significantly change in the other tissues. The GSH levels were significantly increased in the lungs and stomach of rats treated with ABA without any change in the tissues of rats treated with GA(3). Antioxidant enzyme activities such as SOD significantly increased in the spleen of rats treated with ABA and GA(3). Meanwhile, SOD significantly increased in the kidney of rats treated with GA. CAT significantly decreased in the lungs treated with ABA but did not change significantly in all the rest of rat tissues treated with both the PGRs. On the other hand, the ancillary enzyme GR activity decreased in the spleen and increased in the kidney with GA(3) treatment. The drug metabolizing enzyme GST activity significantly decreased in the heart of rats treated with GA(3) but increased in the spleen and lungs of rats treated with both PGRs. The observations presented led us to conclude that administration of subacute ABA and GA(3) promotes lipid peroxidatin content and alters in the antioxidative systems in the rat's various tissues. These data, along with changes, suggest that the PGRs produced oxidative stress in rats during the period of a 25-day subchronic exposure.

Transcriptional profiling of imbibed Brassica napus seed.

Using an Arabidopsis microarray, we compared gene expression between germinating Brassica napus seeds and seeds in which germination was inhibited either by polyethylene glycol (PEG) or by the abscisic acid (ABA) analog PBI429, which produces stronger and longer lasting ABA-like effects. A total of 40 genes were induced relative to the germinating control by both treatments. Conspicuous among these were genes associated with late seed development. We identified 36 genes that were downregulated by both PEG and PBI429. Functions of these genes included carbohydrate metabolism, cell wall-related processes, detoxification of reactive oxygen, and triacylglycerol breakdown. The PBI429 treatment produced an increase in endogenous ABA and increased ABA catabolism. However, PEG treatment did not result in similar effects. The transcription factor ABI5 was consistently upregulated by both treatments and PKL was downregulated. These results suggest a greater importance of ABA signaling and reduced importance of GA signaling in nongerminating seeds.

Allelopathic potential of Citrus junos fruit waste from food processing industry.

The allelopathic potential of Citrus junos fruit waste after juice extraction was investigated. Aqueous methanol extracts of peel, inside and seeds separated from the fruit waste inhibited the growth of the roots and shoots of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), crabgrass (Digitaria sanguinalis L.), lettuce (Lactuca sativa L.), timothy (Pheleum pratense L.), and ryegrass (Lolium multiflorum Lam.). The inhibitory activity of the peel extract was greatest and followed by that of the inside and seed extracts in all bioassays. Significant reductions in the root and shoot growth were observed as the extract concentration was increased. The concentrations of abscisic acid-beta-d-glucopyranosyl ester (ABA-GE) in peel, inside and seeds separated from the C. junos fruit waste were determined, since ABA-GE was found to be one of the main growth inhibitors in C. junos fruit. The concentration was greatest in the peel, followed by the inside and seeds; there was a good correspondence between these concentrations and the inhibitory activities of the extracts. This suggests that ABA-GE may also be involved in the growth inhibitory effect of C. junos waste. These results suggested that C. junos waste may possess allelopathic potential, and the waste may be potentially useful for weed management.