CAT and MDH improve the germination and alleviate the oxidative stress of cryopreserved <Emphasis Type="Italic">Paeonia</Emphasis> and <Emphasis Type="Italic">Magnolia</Emphasis> pollen

Researches have reported that reactive oxygen species (ROS)-induced oxidative stress plays an important role in cell cryodamage during cryopreservation. In the current study, pollen from Magnolia denudata and Paeonia lactiflora ‘Zi Feng Chao Yang’ was cryopreserved and incubated with exogenous catalase (CAT) and malate dehydrogenase (MDH) immediately after thawing. The effect of CAT and MDH on the germination of cryopreserved pollen was measured. Based on that, the ROS level, lipid peroxidation and antioxidants activities in fresh pollen, cryopreserved pollen added with or without CAT or MDH were determined to investigate their relationship with oxidative stress. Pollen from Magnolia and Paeonia showed a significant loss of germination, but marked increase of ROS and malondialdehyde (MDA) production after cryostorage. Antioxidant profiles in them were also enhanced. CAT and MDH addition increased the post-LN pollen germination of Magnolia and Paeonia significantly. Their germination rate achieved the highest with 100 IU ml^?1 MDH and 400 IU ml^?1 CAT application, respectively. Compared to their untreated controls, ROS and MDA accumulation reduced significantly in cryopreserved Magnolia pollen treated with 100 IU ml^?1 MDH, while superoxide dismutase (SOD) activity improved markedly. In the case of Paeonia, significantly lower level of ROS and MDA, but higher activity of CAT and SOD were observed in cryopreserved pollen treated with 400 IU ml^?1 CAT. In conclusion, pollen deterioration after cryopreservation is associated with ROS-induced oxidative stress. Exogenous CAT and MDH can reduce the oxidative damage through the activity stimulation of antioxidant enzymes, and play a protective role in the pollen during cryopreservation.     

Glutathione improves survival of cryopreserved embryogenic calli of <Emphasis Type="Italic">Agapanthus praecox</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis>

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H₂O₂, OH^·, and O ₂ ^·? ) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in  OH^· generation activity and the contents of H₂O₂ and MDA, as well as enhancement in the inhibition of O ₂ ^·? generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.     

Effects of ALA on Photosynthesis,Antioxidant Enzyme Activity,and Gene Expression,and Regulation of Proline Accumulation in Tomato Seedlings Under NaCl Stress

This study examined the effects of 5-aminolevulinic acid (ALA) application on photosynthesis, activity and gene expression of key antioxidant enzymes, and on proline accumulation in tomato (Lycopersicon esculentum Mill. ‘Hezuo 903’) seedlings under NaCl stress. NaCl stress significantly decreased the net photosynthetic rates and inhibited the activity of photosystem II, whereas exogenous ALA application significantly restored the net photosynthetic rates, quantum yield of electron transport, and energy conversion efficiency of photosystem II of tomato under NaCl stress. Production of superoxide, hydrogen peroxide, and malondialdehyde strongly increased in response to NaCl stress, and these increases were significantly counteracted by ALA. ALA increased the activity of reactive oxygen species (ROS) scavenging antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, and upregulated the expression of SOD, APX, and POD, genes that encode these enzymes in NaCl-treated plants. ALA simultaneously increased proline accumulation in tomato seedlings under NaCl stress by regulating the expression of genes that encode ALA biosynthetic enzymes and that control proline biosynthesis and metabolism, for example, expression of GluRS and GluTR was downregulated, accompanied by a significant increase in the expression of P5CS and decline in the expression of ProDH. ALA provided protection against NaCl stress by increasing photosynthetic capacity, regulating antioxidant enzyme gene expression and proline accumulation, and decreasing ROS accumulation and lipid peroxidation in tomato.     

Overexpression of <Emphasis Type="Italic">Populus tomentosa</Emphasis> cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants

Reactive oxygen species (ROS) play key roles in plants and are regulated by several ROS-scavenging enzymes. Ascorbate peroxidase (APX), which catalyzes the reduction of hydrogen peroxide to water, a vital part of ROS formation, plays a significant role in higher plants. In this study, a cytosolic APX gene from Populus tomentosa, named PcAPX, was identified and characterized. Recombinant PcAPX had a calculated mass of 33.24 kD and showed high activity towards ascorbic acid (ASA) and hydrogen peroxide (H₂O₂). Real-time PCR analysis showed that APX mRNA expression levels were higher in leaves than roots or stems of P. tomentosa. Compared with wild-type, transgenic tobacco plants overexpressing PcAPX showed no significant difference in morphology under normal conditions. However, the transgenic plants were more resistant to drought, salt and oxidative stress conditions, as shown by decreased levels of malondialdehyde and increased levels of chlorophyll. Moreover, decreased H₂O₂ levels, increased ASA consumption, an increase in the NADP to NADPH ratio, and higher APX activity in the transgenic plants suggested an increased ability to eliminate ROS. These data suggest that PcAPX overexpression in transgenic tobacco plants can enhance tolerance to drought, salt and oxidative stress. Therefore, APX has a crucial role in abiotic stress tolerance in plants.     

Effects of exogenous <Emphasis Type="Italic">myo</Emphasis>-inositol on leaf water status and oxidative stress of <Emphasis Type="Italic">Capsicum annuum</Emphasis> under drought stress

Drought-stressed plants accumulate cyclitols such as myo-inositol, pinitol, quercitol in the cytosol. These solutes (compatible solutes) protect plants from stress effects. Synthetic myo-inositol was used in the investigation of drought stress tolerance in pepper plants. Hydrogen peroxide (H₂O₂), membrane damage, ascorbate peroxidase (AP), catalase (CAT), proline and calcium increased in plants under drought conditions. Water status, calcium level, glutathione reductase activities increased in myo-inositol treated Capsicum annuum L. (pepper) under drought stress. Exogenous myo-inositol significantly decreased H₂O₂, membrane damage and proline levels and AP (except for 5 µM) and CAT activity, compared with untreated plants. Myo-inositol can play a role as effective as proline in signal transduction and in regulating concentrations of reactive oxygen species within tolerable ranges and in maintaining cell turgor by binding water molecules. Myo-inositol may become a useful instrument to eliminate the negative effects of drought environments.     

The <Emphasis Type="Italic">APX4</Emphasis> locus regulates seed vigor and seedling growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The amino acid sequence of APX4 is similar to other ascorbate peroxidases (APXs), a group of proteins that protect plants from oxidative damage by transferring electrons from ascorbate to detoxify peroxides. In this study, we characterized two apx4 mutant alleles. Translational fusions with GFP indicated APX4 localizes to chloroplasts. Both apx4 mutant alleles formed chlorotic cotyledons with significantly reduced chlorophyll a, chlorophyll b and lutein. Given the homology of APX to ROS-scavenging proteins, this result is consistent with APX4 protecting seedling photosystems from oxidation. The growth of apx4 seedlings was stunted early in seedling development. In addition, APX4 altered seed quality by affecting seed coat formation. While apx4 seed development appeared normal, the seed coat was darker and more permeable than the wild type. In addition, accelerated aging tests showed that apx4 seeds were more sensitive to environmental stress than the wild-type seeds. If APX4 affects seed pigment biosynthesis or reduction, the seed coat color and permeability phenotypes are explained. apx4 mutants had cotyledon chlorosis, increased H₂O₂ accumulation, and reduced soluble APX activity in seedlings. These results indicate that APX4 is involved in the ROS-scavenging process in chloroplasts.     

Aminolevulinic acid and nitric oxide regulate oxidative defense and secondary metabolisms in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under drought stress

To minimize the damaging effects of stresses, plant growth regulators (PGRs) are widely used to sustain the plant life under stress-prone environments. So, a study was carried out to evaluate the response of two canola (Brassica napus L.) cultivars, Dunkeld and Cyclone, to foliar-applied two potential PGRs, nitric oxide (NO) and 5-aminolevulinic acid, under water deficit conditions. In this study, the levels of NO and ALA used were 0.02 and 0.895 mM, respectively. Plants of both canola cultivars were subjected to control (100% field capacity) and water deficit (60% field capacity). Drought stress significantly decreased growth, chlorophyll pigments, relative water contents (RWC), and soluble proteins, while it increased relative membrane permeability (RMP), proline, glycinebetaine (GB), malondialdehyde (MDA), total phenolics, and activities of catalase (CAT) and peroxidase (POD) enzymes in both cultivars. Foliar application of PGRs improved growth, chlorophyll a, GB, total phenolics, CAT activity, and total soluble proteins, while it decreased RMP, MDA, and POD activity in both canola cultivars. Other physio-biochemical attributes such as chlorophyll b, RWC, hydrogen peroxide (H₂O₂) and proline contents as well as superoxide dismutase (SOD) activity remained unaffected due to application of PGRs. So, the results of the present study suggest that exogenous application of NO and ALA could be useful to enhance the drought tolerance of canola plants by up-regulating the oxidative defense system, osmoprotectant accumulation, and minimizing the lipid peroxidation.     

Autotoxicity in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seedlings is alleviated by silicon through an increase in the activity of antioxidant enzymes and by mitigating lipid peroxidation

Autotoxicity in plants limits their growth and that of nearby plants of the same species, which has obvious implications in crop yield and quality. Silicon (Si) has been shown to increase plant tolerance to autotoxic stress. However, the physiological mechanisms underlying the effects of Si in alleviating autotoxicity during germination in cucumber (Cucumis sativus L.) are unknown. Cinnamic acid derivatives, such as 3-phenylpropionic acid (PA), are a class of autotoxins present in cucumber root exudates. Our objective was to investigate Si-induced autotoxic stress tolerance in cucumber seedlings by focusing on the effects of Si on the induction of antioxidant defense pathways. We found that PA treatment significantly reduced seed germination, radicle length, lateral root number, fresh weight, AsA and GSH contents, and the activities of SOD, CAT, and APX in cucumber seedlings, while it increased membrane permeability and levels of MDA, proline, O₂^-, and H₂O₂. Application of Si enhanced growth of PA-treated plants and significantly increased germination rate, radicle length, lateral root number, fresh weight, AsA and GSH levels, and SOD, CAT, POD, and APX activities. These results suggest that exogenous Si alleviates autotoxicity caused by PA during seed germination by increasing antioxidant enzyme activities and mitigating lipid peroxidation.     

Impairment of <Emphasis Type="Italic">ntc</Emphasis>A gene revealed its role in regulating iron homeostasis,ROS production and cellular phenotype under iron deficiency in cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120

Iron deficiency ends up into several unavoidable consequences including damaging oxidative stress in cyanobacteria. NtcA is a global nitrogen regulator controls wide range of metabolisms in addition to regulation of nitrogen metabolism. In present communication, NtcA based regulation of iron homeostasis, ROS production and cellular phenotype under iron deficiency in Anabaena 7120 has been investigated. NtcA regulates the concentration dependent iron uptake by controlling the expression of furA gene. NtcA also regulated pigment synthesis and phenotypic alterations in Anabaena 7120. A significant increase in ROS production and corresponding reduction in the activities of antioxidative enzymes (SOD, CAT, APX and GR) in CSE2 mutant strain in contrast to wild type Anabaena 7120 also suggested the possible involvement of NtcA in protection against oxidative stress in iron deficiency. NtcA has no impact on the expression of furB and furC in spite of presence of consensus NtcA binding site (NBS) and ?10 boxes in their promoter. NtcA also regulates the thylakoid arrangement as well as related photosynthetic and respiration rates under iron deficiency in Anabaena 7120. Overall results suggested that NtcA regulates iron acquisition and in turn protect Anabaena cells from the damaging effects of oxidative stress induced under iron deficiency.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.