Cytosolic Ascorbate Peroxidase Research Articles

Integrated omics approach reveals the molecular pathways activated in tomato by Kocuria rhizophila, a soil plant growth-promoting bacterium

Plant microbial biostimulants application has become a promising and eco-friendly agricultural strategy to improve crop yields, reducing chemical inputs for more sustainable cropping systems. The soil dwelling bacterium Kocuria rhizophila was previously characterized as Plant Growth Promoting Bacteria (PGPB) for its multiple PGP traits, such as indole-3-acetic acid production, phosphate solubilization capability and salt and drought stress tolerance. Here, we evaluated by a multi-omics approach, the PGP activity of K. rhizophila on tomato, revealing the molecular pathways by which it promotes plant growth. Transcriptomic analysis showed several up-regulated genes mainly related to amino acid metabolism, cell wall organization, lipid and secondary metabolism, together with a modulation in the DNA methylation profile, after PGPB inoculation. In agreement, proteins involved in photosynthesis, cell division, and plant growth were highly accumulated by K. rhizophila. Furthermore, “amino acid and peptides”, “monosaccharides”, and “TCA” classes of metabolites resulted the most affected by PGPB treatment, as well as dopamine, a catecholamine neurotransmitter mediating plant growth through S-adenosylmethionine decarboxylase (SAMDC), a gene enhancing the vegetative growth, up-regulated in tomato by K. rhizophila treatment. Interestingly, eight gene modules well correlated with differentially accumulated proteins (DAPs) and metabolites (DAMs), among which two modules showed the highest correlation with nine proteins, including a nucleoside diphosphate kinase, and cytosolic ascorbate peroxidase, as well as with several amino acids and metabolites involved in TCA cycle. Overall, our findings highlighted that sugars and amino acids, energy regulators, involved in tomato plant growth, were strongly modulated by the K. rhizophila-plant interaction.

Millmeter-wave irradiation regulates mRNA-expression and the ubiquitin-proteasome system in wheat exposed to flooding stress

The irradiation with millimeter-wave (MMW) of wheat seeds promotes root growth under flooding stress; however, its role is not completely clarified. Nuclear proteomics was performed, to reveal the role of MMW irradiation in enhancing flooding tolerance. The purity of nuclear fractions purified from roots was verified. Histone, which is a protein marker for nuclear-purification efficiency, was enriched; and cytosolic ascorbate peroxidase was reduced in the nuclear fraction. The principal-component analysis of proteome displayed that the irradiation of seeds affected nuclear proteins in roots grown under flooding stress. Proteins detected using proteomic analysis were verified using immunoblot analysis. Histone H3 accumulated under flooding stress; however, it decreased to the control level by irradiation. Whereas the ubiquitin accumulated in roots grown under stress when seeds were irradiated. These results suggest that MMW irradiation improves wheat-root growth under flooding stress through the regulation of mRNA-expression level and the ubiquitin-proteasome system. SignificanceTo reveal the role of millimeter-wave irradiation in enhancing flooding tolerance in wheat, nuclear proteomics was performed. The principal-component analysis of proteome displayed that irradiation of seeds affected nuclear proteins in roots grown under flooding stress. Proteins detected using proteomic analysis were verified using immunoblot analysis. Histone H3 accumulated under flooding stress; however, it decreased to the control level with irradiation. Whereas the ubiquitin accumulated in roots grown under stress when seeds were irradiated. These results suggest that millimeter-wave irradiation improves wheat-root growth under flooding stress through the regulation of mRNA-expression level and the ubiquitin-proteasome system.

Activity of Cytosolic Ascorbate Peroxidase (APX) from Panicum virgatum against Ascorbate and Phenylpropanoids.

APX is a key antioxidant enzyme in higher plants, scavenging H2O2 with ascorbate in several cellular compartments. Here, we report the crystal structures of cytosolic ascorbate peroxidase from switchgrass (Panicum virgatum L., Pvi), a strategic feedstock plant with several end uses. The overall structure of PviAPX was similar to the structures of other APX family members, with a bound ascorbate molecule at the ɣ-heme edge pocket as in other APXs. Our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Significantly, our study suggested that PviAPX can oxidize a broad range of phenylpropanoids with δ-meso site in a rather similar efficiency, which reflects its role in the fortification of cell walls in response to insect feeding. Based on detailed structural and kinetic analyses and molecular docking, as well as that of closely related APX enzymes, the critical residues in each substrate-binding site of PviAPX are proposed. Taken together, these observations shed new light on the function and catalysis of PviAPX, and potentially benefit efforts improve plant health and biomass quality in bioenergy and forage crops.

A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids.

In planta, H2O2 is produced as a by-product of enzymatic reactions and during defense responses. Ascorbate peroxidase (APX) is a key enzyme involved in scavenging cytotoxic H2O2. Here, we report the crystal structure of cytosolic APX from sorghum (Sorghum bicolor) (Sobic.001G410200). While the overall structure of SbAPX was similar to that of other APXs, SbAPX uniquely displayed four bound ascorbates rather than one. In addition to the ɣ-heme pocket identified in other APXs, ascorbates were bound at the δ-meso and two solvent-exposed pockets. Consistent with the presence of multiple binding sites, our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Bound ascorbate at two surface sites established an intricate proton network with ascorbate at the ɣ-heme edge and δ-meso sites. Based on crystal structures, steady-state kinetics, and site-directed mutagenesis results, both ascorbate molecules at the ɣ-heme edge and the one at the surface are expected to participate in the oxidation reaction. We provide evidence that the H2O2-dependent oxidation of ascorbate by APX produces a C2-hydrated bicyclic hemiketal form of dehydroascorbic acid at the ɣ-heme edge, indicating two successive electron transfers from a single-bound ascorbate. In addition, the δ-meso site was shared with several organic compounds, including p-coumaric acid and other phenylpropanoids, for the potential radicalization reaction. Site-directed mutagenesis of the critical residue at the ɣ-heme edge (R172A) only partially reduced polymerization activity. Thus, APX removes stress-generated H2O2 with ascorbates, and also uses this same H2O2 to potentially fortify cell walls via oxidative polymerization of phenylpropanoids in response to stress.

Ascorbate peroxidase 1 allows monitoring of cytosolic accumulation of effector-triggered reactive oxygen species using a luminol-based assay.

Biphasic production of reactive oxygen species (ROS) has been observed in plants treated with avirulent bacterial strains. The first transient peak corresponds to pattern-triggered immunity (PTI)-ROS, whereas the second long-lasting peak corresponds to effector-triggered immunity (ETI)-ROS. PTI-ROS are produced in the apoplast by plasma membrane-localized NADPH oxidases, and the recognition of an avirulent effector increases the PTI-ROS regulatory module, leading to ETI-ROS accumulation in the apoplast. However, how apoplastic ETI-ROS signaling is relayed to the cytosol is still unknown. Here, we found that in the absence of cytosolic ascorbate peroxidase 1 (APX1), the second phase of ETI-ROS accumulation was undetectable in Arabidopsis (Arabidopsis thaliana) using luminol-based assays. In addition to being a scavenger of cytosolic H2O2, we discovered that APX1 served as a catalyst in this chemiluminescence ROS assay by employing luminol as an electron donor. A horseradish peroxidase (HRP)-mimicking APX1 mutation (APX1W41F) further enhanced its catalytic activity toward luminol, whereas an HRP-dead APX1 mutation (APX1R38H) reduced its luminol oxidation activity. The cytosolic localization of APX1 implies that ETI-ROS might accumulate in the cytosol. When ROS were detected using a fluorescent dye, green fluorescence was observed in the cytosol 6 h after infiltration with an avirulent bacterial strain. Collectively, these results indicate that ETI-ROS eventually accumulate in the cytosol, and cytosolic APX1 catalyzes luminol oxidation and allows monitoring of the kinetics of ETI-ROS in the cytosol. Our study provides important insights into the spatial dynamics of ROS accumulation in plant immunity.

Kiwifruit (Actinidia chinensis ‘Hongyang’) cytosolic ascorbate peroxidases (AcAPX1 and AcAPX2) enhance salinity tolerance in Arabidopsis thaliana

Ascorbate peroxidase (APX) plays a key role in scavenging reactive oxygen species (ROS) in higher plants. However, there is very little information available on the APXs in kiwifruit (Actinidia), which is an economically and nutritionally important horticultural crop with exceptionally high ascorbic acid (AsA) accumulation. This study aims to identify and characterize two cytosolic APX genes (AcAPX1 and AcAPX2) derived from A. chinensis ‘Hongyang’. The constitutive expression pattern was determined for both AcAPX1 and AcAPX2, and showed relatively higher expression abundances of AcAPX1 in leaf and AcAPX2 in root. Transcript levels of AcAPX1 and AcAPX2 were increased in kiwifruit roots treated with NaCl. Subcellular localization assays using GFP-fusion proteins in Arabidopsis protoplasts showed that both AcAPX1 and AcAPX2 are targeted to the cytosol. Recombinant AcAPX1 or AcAPX2 proteins were successfully expressed in the prokaryotic expression system and their individual ascorbate peroxidase activities were determined. Finally, constitutive over-expression of AcAPX1 or AcAPX2 could dramatically increase total AsA, glutathione level and salinity tolerance under NaCl stress in Arabidopsis thaliana. Our findings revealed that cytosolic AcAPX1/2 may play an important protective role in the responses to unfavorable environmental stimuli in kiwifruit.

CfAPX, a cytosolic ascorbate peroxidase gene from Cryptomeria fortunei, confers tolerance to abiotic stress in transgenic Arabidopsis

Plants subjected to biotic or abiotic stresses produce a large amount of reactive oxygen species (ROS). If ROS cannot be cleared in time, they cause a series of harmful reactions in plants. Ascorbate peroxidase (APX) is a key enzyme that removes ROS from plant cells and plays a vital role in plant stress resistance. However, to date, no studies on APX homologs in Cryptomeria fortunei have been reported. In this study, we isolated complementary DNA (cDNA) encoding APXfrom C. fortunei needles, which is referred to as CfAPX, by rapid amplification of cDNA ends (RACE). The full-length CfAPX sequence was 1226 bp in length and included a 750-bp open reading frame (ORF) encoding a protein of 249 amino acids. Phylogenetic analysis showed that APXs of different plant species have been highly evolutionarily conserved. CfAPX was shown to belong to the cytoplasmic subgroup and was more closely related to GbAPX of the gymnosperm Ginkgo biloba. CfAPX showed no transcriptional activity in yeast cells but was highly expressed in cones. To better handle abiotic stresses, compared with wild-type (WT) Arabidopsis thaliana, 35S::CfAPX transgenic Arabidopsis strongly expressed CfAPX, presented increased antioxidant enzyme activities, ascorbic acid (AsA) contents, chlorophyll levels and fluorescence parameter and reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents. In addition, CfAPX expression in C. fortunei was mostly upregulated under stress. In summary, CfAPX confers abiotic stress responses to plants, which provides a scientific basis for subsequent breeding for increased stress resistance in C. fortunei.

Cross-stress tolerance: Mild nitrogen (N) deficiency effects on drought stress response of tomato (Solanum lycopersicum L.).

Climate change will lead to more frequent and severe drought periods which massively reduce crop production worldwide. Besides drought, nitrogen (N)-deficiency is another critical threat to crop yield production. Drought and N-deficiency both decrease photosynthesis and induce similar adaptive strategies such as longer roots, reduction of biomass, induction of reactive oxygen species (ROS), and antioxidative enzymes. Due to the overlapping response to N-deficiency and drought, understanding the physiological and molecular mechanisms involved in cross-stresses tolerance is crucial for breeding strategies and achieving multiple stress resistance and eventually more sustainable agriculture. The objective of this study was to investigate the effect of a mild N-deficiency on drought stress tolerance of tomato plants (Solanum lycopersicum L., cv. Moneymaker). Various morphological and physiological parameters such as dry biomass, root length, water potential, SPAD values, stomatal conductance, and compatible solutes accumulation (proline and sugar) were analyzed. Moreover, the expression of ROS scavenging marker genes, cytosolic ASCORBATE PEROXIDASES (cAPX1, cAPX2, and cAPX3), were investigated. Our results showed that a former mild N-deficiency (2mM NO3 -) enhances plant adaptive response to drought stress (4days) when compared to the plants treated with adequate N (5mM NO3 -). The improved adaptive response was reflected in higher aboveground biomass, longer root, increased specific leaf weight, enhanced stomatal conductance (without reducing water content), and higher leaf sugar content. Moreover, the APX1gene showed a higher expression level compared to control under N-deficiency and in combination with drought in the leaf, after a one-week recovery period. Our finding highlights a potentially positive link between a former mild N-deficiency and subsequent drought stress response in tomato. Combining the morphological and physiological response with underlying gene regulatory networks under consecutive stress, provide a powerful tool for improving multiple stress resistance in tomato which can be further transferred to other economically important crops.

RBOH1-dependent H2O2 mediates spermine-induced antioxidant enzyme system to enhance tomato seedling tolerance to salinity–alkalinity stress

Salinity–alkalinity stress is a limiting factor in tomato production in the world. Plants perceive salinity–alkalinity stress by activating signaling pathways to increase plant tolerance (Xu et al., 2020). Here, we investigated whether spermine (Spm) induces respiratory burst oxidase homolog 1 (RBOH1) and hydrogen peroxide (H2O2) signaling in response to salinity–alkalinity stress in tomato. The results showed that exogenous Spm induced the expression of RBOH1 and the accumulation of H2O2 under normal condition. Accordingly, we tested the function of H2O2 signal in tomato seedlings and found that exogenous H2O2 increased the expression levels of Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase 1 (CAT1), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of SOD (EC 1.15.1.1), CAT (EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), and GR (EC 1.6.4.2) in tomato seedlings under salinity–alkalinity stress. DMTU increased the malondialdehyde (MDA) content and relative electrical conductivity, and the relative water content (RWC), and accelerated leaf yellowing in tomato seedlings under salinity–alkalinity stress, even though we sprayed Spm on tomato leaves. We also found that RBOH1 silencing decreased the expression levels of Cu/Zn-SOD, CAT1, cAPX, and GR1 and the activities of SOD, CAT, APX, and GR when tomato seedlings were under salinity–alkalinity stress. Exogenous Spm did not increase RWC and decrease MDA content in RBOH1 silencing tomato seedlings under salinity–alkalinity stress.

Characterization and expression analysis of ascorbate peroxidase from the moss Dicranum scoparium during abiotic stresses

Ascorbate peroxidase (APX) is an important antioxidant enzyme responsible for the conversion of H2O2 to H2O and O2. In this study, APX was studied in the widespread boreal cushion moss Dicranum scoparium. Native PAGE of crude extracts of moss thalli revealed the presence of APX isoforms in D. scoparium with a range of molecular masses. An APX complementary DNA (cDNA) gene of 771-bp length was cloned and designated as DsAPX. The cloned coding domain sequence (CDS) encoded a 256 amino acid polypeptide, and the predicted protein product was calculated to have a molecular mass of 28.4 kDa with an isoelectric point of 5.7. Several highly conserved sites important for enzyme activity were predicted using bioinformatic tools. The DsAPX protein has similarities of 91%, 78%, 67% and 66% with APX homologs from Grimmia pilifera, Physcomitrella patens, Zea mays and Nicotiana tomentosiformis, respectively. The high homology with cytosolic APX from G. pilifera strongly suggests that the cloned DsAPX gene encodes a cytosolic APX. We studied the role of APX in the tolerance of this moss to abiotic stresses. Changes in both APX activity and DsAPX gene expression, estimated using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), showed that the gene was up-regulated in response to desiccation/rehydration, and expression was maintained during a heat stress of +50°C. By contrast, DsAPX was down-regulated by a freezing (–20°C) treatment. Results obtained extend our knowledge of the diversity of APX isoforms that occur in bryophytes and suggest that APX may be involved in the tolerance of D. scoparium to abiotic stresses.

Seed inoculation of desert-plant growth-promoting rhizobacteria induce biochemical alterations and develop resistance against water stress in wheat.

Water shortage limits agricultural productivity, so strategies to get higher yields in dry agricultural systems is vital to circumvent the effect of climate change and land-shortage. The plant rhizosphere harbors beneficial bacteria able to confer biotic/abiotic tolerance along with a positive impact on plant growth. Herein, three bacterial strains, Proteus mirabilis R2, Pseudomonas balearica RF-2 and Cronobacter sakazakii RF-4 (accessions: LS975374, LS975373, LS975370, respectively) isolated from native desert-weeds were investigated for their response to improve wheat growth under drought stress. The bacteria showed drought tolerance up to 20% polyethylene glycol (PEG; -0.6MPa), and salt (65-97 g l-1 ), 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity, P/Zn/K-solubilization, calcite degradation, IAA, and siderophore production. The plant growth-promoting rhizobacteria (PGPR) were evaluated on wheat under water stress. The P. balearica strain RF-2 primed seeds showed a maximum promptness index and germination index under PEG-stress, that is, 68% and 100%, respectively. Inoculation significantly improved plant growth, leaf area, and biomass under water stress. P. mirabilis R2 inoculated plant leaves showed the highest water contents as compared to the plants inoculated with other strains. C. sakazakii RF-4 inoculated plants showed minimum cell injury, electrolyte leakage, and maximum cell membrane stability at PEG stress. After 13 days exposure to drought, C. sakazakii RF-4 treated plants showed an overall higher expression of cytosolic ascorbate peroxidase (cAPX) and ribulose-bisphosphate carboxylase (rbcL) genes. The activity of stress-induced catalase and polyphenol oxidase was reduced, while that of peroxidase and superoxide dismutase increased after inoculation but the response was temporal. Taken together, this data explains that different PGPR (especially C. sakazakii RF-4) modulate differential responses in wheat that eventually leads towards drought tolerance, hence, it has the potential to enhance crop production in arid regions.

ASCORBATE PEROXIDASE6 delays the onset of age-dependent leaf senescence.

Age-dependent changes in reactive oxygen species (ROS) levels are critical in leaf senescence. While H2O2-reducing enzymes such as catalases and cytosolic ASCORBATE PEROXIDASE1 (APX1) tightly control the oxidative load during senescence, their regulation and function are not specific to senescence. Previously, we identified the role of ASCORBATE PEROXIDASE6 (APX6) during seed maturation in Arabidopsis (Arabidopsis thaliana). Here, we show that APX6 is a bona fide senescence-associated gene. APX6 expression is specifically induced in aging leaves and in response to senescence-promoting stimuli such as abscisic acid (ABA), extended darkness, and osmotic stress. apx6 mutants showed early developmental senescence and increased sensitivity to dark stress. Reduced APX activity, increased H2O2 level, and altered redox state of the ascorbate pool in mature pre-senescing green leaves of the apx6 mutants correlated with the early onset of senescence. Using transient expression assays in Nicotiana benthamiana leaves, we unraveled the age-dependent post-transcriptional regulation of APX6. We then identified the coding sequence of APX6 as a potential target of miR398, which is a key regulator of copper redistribution. Furthermore, we showed that mutants of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), the master regulator of copper homeostasis and miR398 expression, have a higher APX6 level compared with the wild type, which further increased under copper deficiency. Our study suggests that APX6 is a modulator of ROS/redox homeostasis and signaling in aging leaves that plays an important role in developmental- and stress-induced senescence programs.

細胞質型アスコルビン酸ペルオキシダーゼは4-クマル酸3-ヒドロキシラーゼとしてリグニン合成に関与する

細胞質型アスコルビン酸ペルオキシダーゼは4-クマル酸3-ヒドロキシラーゼとしてリグニン合成に関与する

Cytosolic ascorbate peroxidase 1 modulates ascorbic acid metabolism through cooperating with nitrogen regulatory protein P-II in tea plant under nitrogen deficiency stress

Nitrogen (N) element is essential nutrient, and affect metabolism of secondary metabolites in higher plants. Ascorbate peroxidase (APX) plays an important role in ascorbic acid (AsA) metabolism of tea plant. However, the roles of cytosolic ascorbate peroxidase 1 (CsAPX1) in AsA metabolism under N deficiency stress in tea plant remains unclear in detail. In this work, nitrogen regulatory protein P-II (CsGLB1) and CsAPX1 were identified by isobaric tags for relative and absolute quantitation (iTRAQ) from tea plant. The cell growth rates in transgenic Escherichia coli overexpressing CsAPX1 and CsGLB1 were higher than empty vector under N sufficiency condition. Phenotype of shoots and roots, AsA accumulation, and expression levels of AtAPX1 and AtGLB1 genes were changed in transgenic Arabidopsis hosting CsAPX1 under N deficiency stress. These findings suggested that cytosolic CsAPX1 acted a regulator in AsA accumulation through cooperating with GLB1 under N deficiency stress in tea plant.

Cytosolic Ascorbate Peroxidases Plays a Critical Role in Photosynthesis by Modulating Reactive Oxygen Species Level in Stomatal Guard Cell

Photosynthetic rate is one of the key factors limiting yield of cotton. Reactive oxygen species (ROS) generated by abiotic stress imposes numerous detrimental effects and causes tremendous loss of yield. It is worth to study whether ROS scavenging enzymes could affect yield through regulating photosynthetic rate in cotton. In this study, we created transgenic cotton with changes of endogenous ROS by overexpressing or suppressing the expression of cytosolic ascorbate peroxidases (APXs), which are hydrogen peroxide (H2O2) scavenging enzymes in plants. The suppression of cytosolic APXs by RNAi brings about a great influence on plant growth and development. Plant height and leaf size declined, and yield-related traits including single boll weight, seed weight, seed size, and lint weight dropped significantly, in IAO lines (cytosolic APX-suppressed lines). The stunted plant growth was due to the decrease of plant photosynthetic rate. The evidences showed that increased ROS level in guard cells inhibited stomatal opening and suppressed the absorption of CO2 and H2O in IAO line. The decrease of water content and the increase of water loss rate in leaf exacerbated the decline of photosynthetic rate in cytosolic APX-suppressed lines. Based on these results, it implies that cytosolic APXs as a whole play an important role in maintaining REDOX balance to regulate photosynthetic rate and yield in cotton.

PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in Populus.

ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) plays an important role in stress responses, but the transcriptional regulation of ZAT12 in response to abiotic stress remains unclear. In this study, we confirmed that a SALT TOLERANCE ZINC FINGER1 transcription factor from Populus euphratica (PeSTZ1) could regulate the expression of PeZAT12 by dual-luciferase reporter (DLR) assay and electrophoretic mobility shift assay. The expression of PeSTZ1 was rapidly induced by NaCl and hydrogen peroxide (H2O2) treatments. Overexpressing PeSTZ1 in poplar 84K (Populus alba × Populus glandulosa) plant was endowed with a strong tolerance to salt stress. Under salt stress, transgenic poplar exhibited higher expression levels of PeZAT12 and accumulated a larger amount of antioxidant than the wild-type plants. Meanwhile, ASCORBATE PEROXIDASE2 (PeAPX2) can be activated by PeZAT12 and PeSTZ1, promoting the accumulation of cytosolic ascorbate peroxidase (APX) to scavenge reactive oxygen species (ROS) under salt stress. This new regulatory model (PeSTZ1-PeZAT12-PeAPX2) was found in poplar, providing a new idea and insight for the interpretation of poplar resistance. Transgenic poplar reduced the accumulation of ROS, restrained the degradation of chlorophyll and guaranteed the photosynthesis and electron transport system. On the other hand, transgenic poplar slickly adjusted K+/Na+ homeostasis to alleviate salt toxicity in photosynthetic organs of plants under salt stress and then increased biomass accumulation. In summary, PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in poplar.

The role and proteomic analysis of ethylene in hydrogen gas-induced adventitious rooting development in cucumber (Cucumis sativus L.) explants.

Previous studies have shown that both hydrogen gas (H2) and ethylene (ETH) play positive roles in plant adventitious rooting. However, the relationship between H2and ETH during this process has not been explored and remains insufficiently understood. In this study, cucumber (Cucumis sativus L.) was used to explore the proteomic changes in ETH-H2-induced rooting. Our results show that hydrogen-rich water (HRW) and ethylene-releasing compound (ethephon) at proper concentrations promote adventitious rooting, with maximal biological responses occurring at 50% HRW or 0.5 µM ethephon. ETH inhibitors aminoethoxyvinylglycine (AVG) and AgNO3 cause partial inhibition of adventitious rooting induced by H2, suggesting that ETH might be involved in H2-induced adventitious rooting. According to two-dimensional electrophoresis (2-DE) and mass spectrometric analyses, compared with the control, 9 proteins were up-regulated while 15 proteins were down-regulated in HRW treatment; four proteins were up-regulated while 10 proteins were down-regulated in ethephon treatment; and one protein was up-regulated while nine proteins were down-regulated in HRW+AVG treatment. Six of these differentially accumulated proteins were further analyzed, including photosynthesis -related proteins (ribulose-1,5-bisphosphate carall boxylase smsubunit (Rubisco), sedoheptulose-1,7-bisphosphatase (SBPase), oxygen-evolving enhancer protein (OEE1)), amino and metabolism-related protein (threonine dehydratase (TDH)), stress response-related protein (cytosolic ascorbate peroxidase (CAPX)), and folding, modification and degradation-related protein (protein disulfide-isomerase (PDI)). Moreover, the results of real-time PCR about the mRNA levels of these genes in various treatments were consistent with the 2-DE results. Therefore, ETH may be the downstream signaling molecule during H2- induced adventitious rooting and proteins Rubisco, SBPase, OEE1, TDH, CAPX and PDI may play important roles during the process.

Ectopic overexpression of cytosolic ascorbate peroxidase gene (Apx1) improves salinity stress tolerance in Brassica juncea by strengthening antioxidative defense mechanism

Salinity stress is considered to be a key constrain that reduces the crop productivity by impairing plant growth and development. During salt stress condition, an underlying mechanism for reduction in crop yield is increase in ROS level that can potentially harm cellular macromolecules, leading to disruption of essential physiological and biochemical processes. Plants possess a complex antioxidative defense machinery for scavenging these ROS. Ascorbate peroxidase (APX, E.C. 1.11.1.11), is a crucial antioxidant enzyme involved in Ascorbate–Glutathione pathway that primarily detoxifies the negative impact of H2O2 in cell. The efficient scavenging of H2O2 is a prerequisite for enhanced tolerance to salinity stress. Here, we have inspected whether over-expression of APX could provide protection against salinity stress. Cytosolic ascorbate peroxidase (Apx1) gene, isolated from Arabidopsis thaliana, was chosen as the candidate gene for strengthening the antioxidative defense system of Brassica juncea. Physiological parameters were employed to analyze the growth status of transgenic plants. Leaf disc assay was done to evaluate the salinity stress tolerance potential of transgenic plants, using several physiological and biochemical parameters. Under salinity stress, the transgenic plants performed well as compared to their non-transgenic counterparts; as revealed through greater proline accumulation, increased chlorophyll stability index, lower chlorophyll a/b ratio, and higher antioxidative enzyme activities. Further, the lower H2O2 levels were well correlated with lesser membrane damage as measured through MDA content. Collectively, our results clearly depicted that ectopic overexpression of AtApx1 gene was able to confer salinity stress tolerance by strengthening the antioxidative defense system in B. juncea.

Cloning and overexpression of the ascorbate peroxidase gene from the yam (Dioscorea alata) enhances chilling and flood tolerance in transgenic Arabidopsis.

Minghuai 1 (MH1) is a yam (Dioscorea alata) cultivar with high tolerance to flooding but sensitivity to chilling. MH1 responded differently to chilling and flooding according to various physiological parameters and antioxidant enzymes. Flooding led to an increase in ascorbate peroxidase (APX) activity in both roots and leaves, while chilling did not affect APX activity. The full length DaAPX ORF sequence from MH1 (750bp) was then cloned. Phylogenetic analysis showed that plant cytosolic APXs into four major clusters and DaAPX was closely related to Oncidium. The DaAPX gene driven by a 35S promoter was transferred into Arabidopsis. The gene expression and enzyme activity of APX in the DaAPX transgenic lines 1-3 were significantly higher than in wild type (WT) plants. Compared to WT plants, seedling growth characteristics were significantly better in all transgenic lines under chilling, flooding, and oxidative stresses, indicating that the overexpression of DaAPX in Arabidopsis enhanced tolerance to several abiotic stresses. MH1 plants supplied with H2O2 presented an increase in the activity of APX leading to enhanced tolerance to chilling. Functional characterization of the APX gene should improve our understanding of the chilling- and flood-response mechanism in the yam.

Groundnut AhcAPX conferred abiotic stress tolerance in transgenic banana through modulation of the ascorbate-glutathione pathway.

A stress inducible cytosolic ascorbate peroxidase gene (AhcAPX) was ectopically expressed in banana (cv. Grand naine) plants to strengthen their antioxidant capacity. High level of AhcAPX gene transcripts and enzyme suggested constitutive and functional expression of candidate gene in transgenic (TR) plants. The tolerance level of in vitro and in vivo grown TR banana plantlets were assessed against salt and drought stress. The TR banana plants conferred tolerance against the abiotic stresses by maintaining a high redox state of ascorbate and glutathione, which correlated with lower accumulation of H2O2, O2 ⋅- and higher level of antioxidant enzyme (SOD, APX, CAT, GR, DHAR and MDHAR) activities. The efficacy of AhcAPX over-expression was also investigated in terms of different physiochemical attributes of TR and untransformed control plants, such as, proline content, membrane stability, electrolyte leakage and chlorophyll retention. The TR plants showed higher photochemical efficiency of PSII (Fv/Fm), and stomatal attributes under photosynthesis generated reactive oxygen species (ROS) stress. The outcome of present investigation suggest that ectopic expression of AhcAPX gene in banana enhances the tolerance to drought and salt stress by annulling the damage caused by ROS.