Investigating Epidermal Growth Factor Receptor Trafficking with High-Resolution Enzymatic Protein-Tagging and Transmission Electron Microscopy.

Enzymatic ascorbate peroxidase (APEX) tagging allows for high-resolution, three-dimensional protein distribution analyses in cells and tissues. This chapter describes the application of APEX-tagging to visualize the trafficking of the epidermal growth factor receptor (EGFR) during epidermal growth factor-mediated receptor activation. Here, we describe the preparation of cells, methods to validate the stimulation of the EGFR, and visualization of the APEX-resolved distribution of the EGFR in the transmission electron microscope.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

5-Aminolevulinic acid treatment mitigates pesticide stress in bean seedlings by regulating stress-related gene expression and retrotransposon movements.

Overdoses of pesticides lead to a decrease in the yield and quality of plants, such as beans. The unconscious use of deltamethrin, one of the synthetic insecticides, increases the amount of reactive oxygen species (ROS) by causing oxidative stress in plants. In this case, plants tolerate stress by activating the antioxidant defense mechanism and many genes. 5-Aminolevulinic acid (ALA) improves tolerance to stress by acting exogenously in low doses. There are many gene families that are effective in the regulation of this mechanism. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. In this study, the expression levels of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and stress-associated protein (SAP) genes were determined by Q-PCR in deltamethrin (0.5 ppm) and various doses (20, 40, and 80 mg/l) of ALA-treated bean seedlings. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. It was determined that deltamethrin increased the expression of SOD (1.8-fold), GPX (1.4-fold), CAT (2.7-fold), and SAP (2.5-fold) genes, while 20 and 40 mg/l ALA gradually increased the expression of these genes at levels close to control, but 80 mg/l ALA increased the expression of these genes almost to the same level as deltamethrin (2.1-fold, 1.4-fold, 2.6-fold, and 2.6-fold in SOD, GPX, CAT, and SAP genes, respectively). In addition, retrotransposon-microsatellite amplified polymorphism (REMAP) was performed to determine the polymorphism caused by retrotransposon movements. While deltamethrin treatment has caused a decrease in genomic template stability (GTS) (27%), ALA treatments have prevented this decline. At doses of 20, 40, and 80 mg/L of ALA treatments, the GTS ratios were determined to be 96.8%, 74.6%, and 58.7%, respectively. Collectively, these findings demonstrated that ALA has the utility of alleviating pesticide stress effects on beans.

Modification in the ascorbate-glutathione cycle leads to a better acclimation to high light in the rose Bengal resistant mutant of Nannochloropsis oceanica.

Understanding how to adapt outdoor cultures of Nannochloropsis oceanica to high light (HL) is vital for boosting productivity. The N. oceanica RB2 mutant, obtained via ethyl methanesulfonate mutagenesis, was chosen for its tolerance to Rose Bengal (RB), a singlet oxygen (1O2) generator. Compared to the wild type (WT), the RB2 mutant showed higher resilience to excess light conditions. Analyzing the ascorbate-glutathione cycle (AGC), involving ascorbate peroxidases (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.8.1.7), in the RB2 mutant under HL stress provided valuable insights. At 250 µmol photon m-2 s-1 (HL), the WT strain displayed superoxide anion radicals (O2▪-) and hydrogen peroxide (H2O2) accumulation, increased lipid peroxidation, and cell death compared to normal light (NL) conditions (50 µmol photon m-2 s-1). The RB2 mutant didn't accumulate O2▪- and H2O2 after HL exposure, and exhibited increased APX, DHAR, and GR activities and transcript levels compared to WT and remained consistent after HL treatment. Although the RB2 mutant had a smaller ascorbate (AsA) pool than the WT, its ability to regenerate dehydroascorbate (DHA) increased post HL exposure, indicated by a higher AsA/DHA ratio. Additionally, under HL conditions, the RB2 mutant displayed an improved glutathione (GSH) regeneration rate (GSH/GSSG ratio) without changing the GSH pool size. Remarkably, H2O2 or menadione (a O2▪- donor) treatment induced cell death in the WT strain but not in the RB2 mutant. These findings emphasize the essential role of AGC in the RB2 mutant of Nannochloropsis in handling photo-oxidative stress.

Graphene oxide-based aerogel stimulates growth, mercury accumulation, photosynthesis-related gene expression, antioxidant efficiency and redox status in wheat under mercury exposure.

Mercury (Hg) pollution is a global concern in cropland systems. Hg contamination causes a disruption in the growth, energy metabolism, redox balance, and photosynthetic activity of plants. In the removal of Hg toxicity, a recent critical strategy is the use of aerogels with biodegradability and biocompatibility. However, it is unknown how graphene oxide-based aerogels stimulate the defense systems in wheat plants exposed to Hg toxicity. Therefore, in this study, the photosynthetic, genetic, and biochemical effects of reduced graphene oxide aerogel treatments (gA; 50-100-250 mg L-1) were examined in wheat (Triticum aestivum) under Hg stress (50 µM HgCl2). The relative growth rate (RGR) significantly decreased (84%) in response to Hg stress. However, the reduced RGR and water relations (RWC) of wheat were improved by gA treatments. The impaired gas exchange levels (stomatal conductance, carbon assimilation rate, intercellular CO2 concentrations, and transpiration rate) caused by stress were reversed under Hg plus gAs. Additionally, stress hampered chlorophyll fluorescence (Fv/Fo, Fv/Fm), and under Hg toxicity the expression of psaA genes was reduced (>0.4-fold), but psaB gene was significantly up-regulated (>3-fold) which are the genes involved in PSI. By increasing expression patterns of both genes relating to PSI, gAs reversed the adverse consequences on Fv/Fo and Fv/Fm in the presence of excessive Hg concentration. The activities of glutathione S-transferase (GST), glutathione reductase (GR), catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) decreased under Hg toxicity. On the other hand, the activities of superoxide dismutase (SOD), APX, GST, and glutathione peroxidase (GPX) increased following gA treatments against stress, leading to the successful elimination of toxic levels of H2O2 and lipid peroxidation (TBARS content) by decreasing the levels by about 30%, and 40%, respectively. By modulating enzyme/non-enzyme activity/contents including the AsA-GSH cycle, gAs contributed to the protection of the cellular redox state. Most important of all, gA applications were able to reduce Hg intake by approximately 66%. Therefore, these results showed that gAs were effective in highly inhibiting Hg uptake and could significantly increase wheat tolerance to toxicity by eliminating Hg-induced oxidative damage and inhibiting metabolic processes involved in photosynthesis. The findings obtained from the study provide a new perspective on the alleviation roles of reduced graphene oxide aerogels as an effective adsorbent for decreasing damages of mercury toxicity in wheat plants.

Bamboo leaf extract treatment alleviates the surface browning of fresh-cut apple by regulating membrane lipid metabolism and antioxidant properties.

BACKGROUND: The effect of bamboo leaf extract (BLE) on controlling the browning of fresh-cut apple stored at 4 °C was investigated. Browning index, H2 O2 content, O2 - production rate, malondialdehyde (MDA) contents, total phenolic content (TPC) and soluble quinone content (SQC), the activities of polyphenol oxidase (PPO), peroxidase (POD), lipoxygenase (LOX), superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), DPPH (2,2-diphenyl-2-picryl-hydrazyl) and ABTS [2,2-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid)] radical scavenging activities, and the expression of genes related to browning were all investigated. RESULTS: BLE effectively alleviated the surface browning of fresh-cut apple, accompanied by a reduction in SQC, LOX activity, H2 O2 , O2 - production rate and MDA accumulation. Furthermore, BLE treatment enhanced the TPC, enzymatic (SOD, CAT, APX and POD) and non-enzymatic antioxidant activities. Principal component analysis and Pearson correlation analysis found the browning inhibition by BLE is not through the reduction of phenolic substrates and PPO activity. CONCLUSION: BLE controls the browning of fresh-cut apple by increasing the antioxidant capacity to scavenge ROS, which could alleviate oxidative damage and maintain the membrane integrity. © 2023 Society of Chemical Industry.

Ethylene-dependent regulation of oxidative stress in the leaves of fusaric acid-treated tomato plants.

The mycotoxin fusaric acid (FA) induces rapid oxidative burst leading to cell death in plants. At the same time, plant defence reactions are mediated by several phytohormones for instance ethylene (ET). However, previously conducted studies leave research gaps on how ET plays a regulatory role under mycotoxin exposure. Therefore, this study aims to the time-dependent effects of two FA concentrations (0.1 mM and 1 mM) were explored on the regulation of reactive oxygen species (ROS) in leaves of wild-type (WT) and ET receptor mutant Never ripe (Nr) tomatoes. FA induced superoxide and H2O2 accumulation in both genotypes in a mycotoxin dose- and exposure time-dependent pattern. 1 mM FA activated NADPH oxidase (+34% compared to the control) and RBOH1 transcript levels in WT leaves. However, superoxide production was significantly higher in Nr with 62% which could contribute to higher lipid peroxidation in this genotype. In parallel, the antioxidative defence mechanisms were also activated. Both peroxidase and superoxide dismutase activities were lower in Nr but ascorbate peroxidase showed one-fold higher activity under 1 mM FA stress than in WT leaves. Interestingly, catalase (CAT) activity decreased upon FA in a time- and concentration-dependent manner and the encoding CAT genes were also downregulated, especially in Nr leaves at 20%. Ascorbate level was decreased and glutathione remained lower in Nr than WT plants under FA exposure. Conclusively, Nr genotype showed more sensitivity to FA-induced ROS suggesting that ET serves defence reactions of plants by activating several enzymatic and non-enzymatic antioxidants to detoxify excess ROS accumulation.

Kinase Inhibitor VvBKI1 Interacts with Ascorbate Peroxidase VvAPX1 Promoting Plant Resistance to Oomycetes.

Downy mildew caused by oomycete pathogen Plasmopara viticola is a devastating disease of grapevine. P. viticola secretes an array of RXLR effectors to enhance virulence. One of these effectors, PvRXLR131, has been reported to interact with grape (Vitis vinifera) BRI1 kinase inhibitor (VvBKI1). BKI1 is conserved in Nicotiana benthamiana and Arabidopsis thaliana. However, the role of VvBKI1 in plant immunity is unknown. Here, we found transient expression of VvBKI1 in grapevine and N. benthamiana increased its resistance to P. viticola and Phytophthora capsici, respectively. Furthermore, ectopic expression of VvBKI1 in Arabidopsis can increase its resistance to downy mildew caused by Hyaloperonospora arabidopsidis. Further experiments revealed that VvBKI1 interacts with a cytoplasmic ascorbate peroxidase, VvAPX1, an ROS-scavenging protein. Transient expression of VvAPX1 in grape and N. benthamiana promoted its resistance against P. viticola, and P. capsici. Moreover, VvAPX1 transgenic Arabidopsis is more resistant to H. arabidopsidis. Furthermore, both VvBKI1 and VvAPX1 transgenic Arabidopsis showed an elevated ascorbate peroxidase activity and enhanced disease resistance. In summary, our findings suggest a positive correlation between APX activity and resistance to oomycetes and that this regulatory network is conserved in V. vinifera, N. benthamiana, and A. thaliana.

Hydrogen sulfide treatment improves quality attributes via regulating the antioxidant system in goji berry (Lycium barbarum L.).

Hydrogen sulfide (H2S) has been identified as a critical gaseous signaling chemical. Herein, the effects of H2S treatment on the postharvest goji berries and antioxidant enzyme activities were determined. H2S application delayed the decay index, loss of firmness, color, flavor, and total sugars and loss of total protein, betaine and ascorbic acid in goji berries during postharvest storage. Meanwhile, H2S noticeably reduced the MDA, H2O2, and O2- accumulation. Additionally, it was shown that H2S increased the activity of catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), glutathione reductase (GR) and superoxide dismutase (SOD) while decreased the quantity of lipoxygenase (LOX). The mRNA expression of LDC, DCD, CAT, APX, POD, GR and SOD was up-regulated but LOX, RBOH-b and RBOH-e was down-regulated in goji berries after H2S treatment. Altogether, H2S could efficiently delay the senescence, improves postharvest quality, increase the bioactive compounds accumulation, and boost the antioxidant capacity of goji berries through modulating antioxidant enzyme system.

Glutamic Acid and Poly-γ-glutamic Acid Enhanced the Heat Resistance of Chinese Cabbage (Brassica rapa L. ssp. pekinensis) by Improving Carotenoid Biosynthesis, Photosynthesis, and ROS Signaling.

Heat stress is one of the most common agrometeorological risks in crop production in the middle and lower reaches of the Yangtze River in China. This study aimed to investigate whether glutamic acid (Glu) or poly-γ-glutamic acid (γ-PGA) biostimulants can improve the thermotolerance of a cool-season Chinese cabbage (Brassica rapa L. ssp. pekinensis) crop. Priming with Glu (2.0 mM) or γ-PGA (20 mg·L-1) was conducted at the third leaf stage by applying as daily foliar sprays for 5 days before 5 days of heat stress (45 °C in 16-h light/35 °C in 8-h dark). Coupled with morpho-physiological and biochemical analyses, transcriptomes of Glu or γ-PGA-primed Chinese cabbage under heat stress were examined by RNA-seq analysis. The results showed that the thermotolerance conferred by Glu and γ-PGA priming was associated with the increased parameters of vegetative growth, gas exchange, and chlorophyll fluorescence. Compared with the control, the dry weights of plants treated with Glu and γ-PGA increased by 51.52% and 39.39%, respectively. Glu and γ-PGA application also significantly increased the contents of total chlorophyll by 42.21% and 23.12%, and carotenoid by 32.00% and 24.00%, respectively. In addition, Glu- and γ-PGA-primed plants markedly inhibited the levels of malondialdehyde, electrolyte leakage, and super-oxide anion radical, which was accompanied by enhanced activity levels of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD). Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) categories within the differentially expressed genes (DEGs) functional clusters of RNA-seq data indicated that the expression levels of the genes for DNA replication, DNA repair system, linoleic acid metabolism, cysteine and methionine metabolism, glutathione metabolism, purine and pyrimidine metabolism, carotenoid biosynthesis, and plant-pathogen interaction were commonly up-regulated by both Glu and γ-PGA priming. Glu treatment enhanced the expression levels of the genes involved in aliphatic glucosinolate and 2-oxocarboxylic acid, while γ-PGA treatment activated carotenoid cleavage reaction to synthesize abscisic acid. Taken together, both Glu and γ-PGA have great potential for the preadaptation of Chinese cabbage seedlings to heat stress, with Glu being more effective than γ-PGA.

Comparative proteomic analysis of wild-type and a SlETR-3 (Nr) mutant reveals an ethylene-induced physiological regulatory network in fresh-cut tomatoes.

Ethylene plays a crucial role in regulating fruit ripening, quality, and defense response. However, the mechanism(s) responsible for wound-induced ethylene regulation of fruit physiology at a network level is unclear. We used mass spectrometry (MS) to identify differences in the physiological response between fresh-cut fruits of wild-type (WT) tomato and an ethylene receptor mutant (SlETR-3) (also referred to as Nr) during storage. We found that Nr mutants exhibited better appearance and quality, as well as higher ethylene levels during the first 3 d of storage at 4 °C. Thirty-seven (0 h), eighty-two (12 h) and twelve (24 h) differentially abundant proteins were identified between the fresh-cut slices of the two genotypes during storage at the designated timepoints. In particular, antioxidant enzymes, such as ascorbate peroxidase, glutathione S-transferase, and peroxiredoxin were highly expressed in WT fruit, which was associated with higher H2O2 production, and high levels of transcription of cell-wall degrading enzymes. Leucine aminopeptidase, a marker enzyme for response to wounding exhibited higher levels in the Nr mutant, which is consistent with its higher production of ethylene. Collectively, our results provide a deeper insight into the ethylene-induced physiological regulatory network that is activated in fresh-cut tomatoes.

Melatonin Enhances Drought Tolerance in Rice Seedlings by Modulating Antioxidant Systems, Osmoregulation, and Corresponding Gene Expression.

Rice is the third largest food crop in the world, especially in Asia. Its production in various regions is affected to different degrees by drought stress. Melatonin (MT), a novel growth regulator, plays an essential role in enhancing stress resistance in crops. Nevertheless, the underlying mechanism by which melatonin helps mitigate drought damage in rice remains unclear. Therefore, in the present study, rice seedlings pretreated with melatonin (200 µM) were stressed with drought (water potential of -0.5 MPa). These rice seedlings were subsequently examined for their phenotypes and physiological and molecular properties, including metabolite contents, enzyme activities, and the corresponding gene expression levels. The findings demonstrated that drought stress induced an increase in malondialdehyde (MDA) levels, lipoxygenase (LOX) activity, and reactive oxygen species (ROS, e.g., O2- and H2O2) in rice seedlings. However, the melatonin application significantly reduced LOX activity and the MDA and ROS contents (O2- production rate and H2O2 content), with a decrease of 29.35%, 47.23%, and (45.54% and 49.33%), respectively. It activated the expression of ALM1, OsPOX1, OsCATC, and OsAPX2, which increased the activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), respectively. Meanwhile, the melatonin pretreatment enhanced the proline, fructose, and sucrose content by inducing OsP5CS, OsSUS7, and OsSPS1 gene expression levels. Moreover, the melatonin pretreatment considerably up-regulated the expression levels of the melatonin synthesis genes TDC2 and ASMT1 under drought stress by 7-fold and 5-fold, approximately. These improvements were reflected by an increase in the relative water content (RWC) and the root-shoot ratio in the drought-stressed rice seedlings that received a melatonin application. Consequently, melatonin considerably reduced the adverse effects of drought stress on rice seedlings and improved rice's ability to tolerate drought by primarily boosting endogenous antioxidant enzymes and osmoregulation abilities.

Multiwalled Carbon Nanotubes Alter the PSII Photochemistry, Photosystem-Related Gene Expressions, and Chloroplastic Antioxidant System in Zea mays under Copper Toxicity.

A critical approach against copper (Cu) toxicity is the use of carbon nanomaterials (CNMs). However, the effect of CNMs on Cu toxicity-exposed chloroplasts is not clear. The photosynthetic, genetic, and biochemical effects of multiwalled carbon nanotubes (50-100-250 mg L-1 CNT) were investigated under Cu stress (50-100 µM CuSO4) in Zea mays chloroplasts. Fv/Fm and Fv/Fo were suppressed under stress. Stress altered the antioxidant system and the expression of psaA, psaB, psbA, and psbD. The chloroplastic activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione S-transferase (GST), and glutathione peroxidase (GPX) increased under CNT + stress, and those of hydrogen peroxide (H2O2) and lipid peroxidation decreased. CNTs were promoted to the maintenance of the redox state by regulating enzyme/non-enzyme activity/contents involved in the AsA-GSH cycle. Furthermore, CNTs inverted the negative effects of Cu by upregulating the transcriptions of photosystem-related genes. However, the high CNT concentration had adverse effects on the antioxidant capacity. CNT has great potential to confer tolerance by reducing Cu-induced damage and protecting the biochemical reactions of photosynthesis.

Effects of exogenous taurine on growth, photosynthesis, oxidative stress, antioxidant enzymes and nutrient accumulation by Trifolium alexandrinum plants under manganese stress.

Plants essentially require manganese (Mn) for their normal metabolic functioning. However, excess Mn in the cellular environment is detrimental to plant growth, development, and physio-biochemical functions. Taurine (TAU) is an amino acid with potent antioxidant and anti-inflammatory properties in animals and humans. However, no previous study has investigated the potential of TAU in plant metal stress tolerance. The current study provides some novel insights into the effect of TAU in modulating the defense system of Trifolium alexandrinum plants under Mn toxicity. Manganese toxicity resulted in higher oxidative stress and membrane damage through increased superoxide radical, hydrogen peroxide, malondialdehyde, and methylglyoxal generation alongside enhanced lipoxygenase (LOX) activity. Mn toxicity also resulted in limited uptake of potassium (K+), phosphorus (P), calcium (Ca2+), and increased the accumulation of Mn in both leaf and roots. However, TAU circumvented the Mn-induced oxidative stress by upregulating the activities of antioxidant enzymes (ascorbate peroxidase, peroxidase, catalase, glutathione reductase, glutathione-S-transferase, and superoxide dismutase) and levels of ascorbic acid, proline, anthocyanins, phenolics, flavonoids and glutathione (GSH). Taurine conspicuously improved the growth, photosynthetic pigments, hydrogen sulphide (H2S), and nitric oxide (NO) levels of Mn stressed plants. Taurine also improved the uptake of K+, Ca2+, P and reduced the Mn content in stressed plants. Overall, exogenous taurine might be a suitable strategy to combat Mn stress in T. alexandrinum plants but applications at field levels for various crops and metal toxicities and economic suitability need to be addressed before final recommendations.

The role of ethylene signalling in the regulation of salt stress response in mature tomato fruits: Metabolism of antioxidants and polyamines.

Salt stress-induced ethylene (ET) can influence the defence responses of plants that can be dependent on plant organs. In this work, the effects of salt stress evoked by 75 mM NaCl treatment were measured in fruits of wild-type (WT) and ET receptor-mutant Never ripe (Nr) tomato. Salt stress reduced the weight and size of fruits both in WT and Nr, which proved to be more pronounced in mutants. In addition, significantly higher H2O2 levels and lipid peroxidation were measured after the salt treatment in Nr as compared to the untreated control than in WT. ET regulated the key antioxidant enzymes, especially ascorbate peroxidase (APX), in WT but in the mutant fruits the activity of APX did not change and the superoxide dismutase and catalase activities were downregulated compared to untreated controls after salt treatment contributing to a higher degree of oxidative stress in Nr fruits. The dependency of PA metabolism on the active ET signalling was investigated for the first time in fruits of Nr mutants under salt stress. 75 mM NaCl enhanced the accumulation of spermine in WT fruits, which was not observed in Nr, but levels of putrescine and spermidine were elevated by salt stress in these tissues. Moreover, the catabolism of PAs was much stronger under high salinity in Nr fruits contributing to higher oxidative stress, which was only partially alleviated by the increased total and reduced ascorbate and glutathione pool. We can conclude that ET-mediated signalling plays a crucial role in the regulation of salt-induced oxidative stress and PA levels in tomato fruits at the mature stage.

Ascorbate peroxidase postcold regulation of chloroplast NADPH dehydrogenase activity controls cold memory.

Exposure of Arabidopsis (Arabidopsis thaliana) to 4°C imprints a cold memory that modulates gene expression in response to a second (triggering) stress stimulus applied several days later. Comparison of plastid transcriptomes of cold-primed and control plants directly before they were exposed to the triggering stimulus showed downregulation of several subunits of chloroplast NADPH dehydrogenase (NDH) and regulatory subunits of ATP synthase. NDH is, like proton gradient 5 (PGR5)-PGR5-like1 (PGRL1), a thylakoid-embedded, ferredoxin-dependent plastoquinone reductase that protects photosystem I and stabilizes ATP synthesis by cyclic electron transport (CET). Like PGRL1A and PGRL1B transcript levels, ndhA and ndhD transcript levels decreased during the 24-h long priming cold treatment. PGRL1 transcript levels were quickly reset in the postcold phase, but expression of ndhA remained low. The transcript abundances of other ndh genes decreased within the next days. Comparison of thylakoid-bound ascorbate peroxidase (tAPX)-free and transiently tAPX-overexpressing or tAPX-downregulating Arabidopsis lines demonstrated that ndh expression is suppressed by postcold induction of tAPX. Four days after cold priming, when tAPX protein accumulation was maximal, NDH activity was almost fully lost. Lack of the NdhH-folding chaperonin Crr27 (Cpn60ß4), but not lack of the NDH activity modulating subunits NdhM, NdhO, or photosynthetic NDH subcomplex B2 (PnsB2), strengthened priming regulation of zinc finger of A. thaliana 10, which is a nuclear-localized target gene of the tAPX-dependent cold-priming pathway. We conclude that cold-priming modifies chloroplast-to-nucleus stress signaling by tAPX-mediated suppression of NDH-dependent CET and that plastid-encoded NdhH, which controls subcomplex A assembly, is of special importance for memory stabilization.

An oligonucleotide/oligosaccharide-binding-fold protein enhances the alternative splicing event producing thylakoid membrane-bound ascorbate peroxidase in Nicotiana tabacum.

The stromal and thylakoid membrane-bound ascorbate peroxidase isoforms are produced by the alternative splicing event of the 3'-terminal region of the APXII gene in spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum), but not in Arabidopsis (Arabidopsis thaliana). However, all alternative splicing variants were detected in APXII gene-transformed Arabidopsis, indicating the occurrence of its regulatory mechanisms in Arabidopsis. The efficiency of this alternative splicing event in producing thylakoid membrane-bound ascorbate peroxidase mRNA is regulated by a splicing regulatory cis element, but trans splicing regulatory factor(s) for alternative splicing remain unclear. To identify this factor, we conducted a forward genetic screen using Arabidopsis in combination with a luciferase reporter system to evaluate the alternative splicing efficiency of thylakoid membrane-bound ascorbate peroxidase mRNA production. We isolated 9 mutant lines that showed low efficiency of the AS in producing thylakoid membrane-bound ascorbate peroxidase mRNA compared with that in the control plants. From one mutant [APXII alternative splicing inhibition (apsi1)], the causal gene responsible for the phenotype, AT5G38890 (oligonucleotide/oligosaccharide-binding-fold protein, APSI1), was identified. The levels of thylakoid membrane-bound ascorbate peroxidase mRNA from the transformed APXII gene decreased and increased in APSI1 knockout and APSI1-overexpressing plants, respectively. APSI1 was localized to the nucleus and specifically bound to the splicing regulatory cis element sequence. Tobacco plants that disrupted the closest homologs of APSI1 showed low levels of endogenous thylakoid membrane-bound ascorbate peroxidase mRNA. These results indicate that APSI1 is an enhancing component of the alternative splicing event of APXII.

Potassium ion regulates hormone, Ca2+ and H2O2 signal transduction and antioxidant activities to improve salt stress resistance in tobacco.

Although improvement of plant salt tolerance by potassium ions (K+) has been widely studied, whether the tolerance is mediated via hormone signaling or antioxidant systems remains to be explored. This study combined plant physiology with transcriptomic techniques to study how K+ interacts with hormones and antioxidant enzymes to improve plant salt tolerance. Tobacco was used as the test material to study the effects of exogenous potassium application on photosynthetic function, hormone signal transduction, and reactive oxygen species (ROS) production under NaCl stress. The study also evaluated the function of the Ca2+ signaling pathway in salt stress tolerance. Transcriptome data showed that 4413 up-regulated genes and 3743 down-regulated genes were found in tobacco leaves treated with NaCl compared with the control. Compared with NaCl, the down-regulated genes in tobacco leaves were significantly reduced under NaCl + KCL treatment. The results showed that NaCl stress caused oxidative damage to tobacco leaves due to increased superoxide anion (O2-) content, superoxide dismutase (SOD) dismutates superoxide anion to produce hydrogen peroxide and the accumulation of H2O2 caused by reduced ascorbate peroxidase (APX) and peroxidase (POD) activities. NaCl stress also increased abscisic acid (ABA) content in tobacco leaves, resulting in stomatal closure and reduced photosynthetic capacity. Transcriptome data showed that 5 SOD, 1 POD, 1 CAT, 5 APX, and 3 GPX genes were significantly down-regulated by the NaCl treatment. Contrarily, NaCl + KCl treatment reduced the accumulation of O2-and SOD activity but increased POD activity, thereby reducing the accumulation of H2O2 and alleviating oxidative damage. The expression of 2 SOD and 3 APX and 2 GPX genes was significantly higher in NaCl + KCl treatment than that in NaCl treatment. Sufficient K+ also increased indole acetic acid (IAA) levels in tobacco leaves under NaCl stress but reduced ABA content, promoting stomatal opening and improving the photosynthetic capacity. In conclusion, K+ can improve plant salt tolerance by alleviating oxidative damage and regulating hormone signal transduction.

Quantitative Measurement of Ascorbate and Glutathione by Spectrophotometry.

Ascorbate and glutathione are key chemical antioxidants present at relatively high concentrations in plant cells. They are also reducing cofactors for enzymes that process hydrogen peroxide in the ascorbate-glutathione pathway. Due to these two related biochemical functions, the compounds form an interface between reactive oxygen species and sensitive cellular components. Therefore, their status can provide reliable and direct information on cell redox state, signaling, and plant health. While several methods exist for quantification of ascorbate and glutathione, simple enzyme-dependent assays allow them to be measured easily and inexpensively in common extracts. This chapter describes a protocol to measure total contents, as well as the major oxidized and reduced forms, of both compounds in plant tissues.

Assessing the effects of nickel on, e.g., Medicago sativa L. nodules using multidisciplinary approach.

Industrial wastes and fertilizers can introduce excessive levels of nickel (Ni) into the environment, potentially causing threats to plants, animals, as well as human beings. However, the number of studies on the effects of Ni toxicity on nodules is fairly limited. To address this issue, the effects of increasing Ni concentration on alfalfa nodules were assessed at chemical, biochemical, and transcriptomic levels. For this purpose, plants were grown in soils supplied with Ni (control, 0 mg/kg; C1, 50 mg/kg; C2, 150 mg/kg; C3, 250 mg/kg; and C4, 500 mg/kg) for 90 days. Ni loads in leaves, roots, and nodules were monitored after the exposure period. A set of biochemical biomarkers of oxidative stress was determined in nodules including antioxidants and metal homeostasis as well as lipid peroxidation. Gene expression levels of the main targets involved in oxidative stress and metal homeostasis were assessed. Our data indicated a high concentration of Ni in leaves, roots, and nodules where values reached 25.64 ± 3.04 mg/kg, 83.23 ± 5.16 mg/kg, and 125.71 ± 4.53 mg/kg in dry weight, respectively. Moreover, a significant increase in nodule biomass was observed in plants exposed to C4 in comparison to control treatment and percentage increased by 63%. Then, lipid peroxidation increased with a rate of 95% in nodules exposed to C4. Enzymatic activities were enhanced remarkably, suggesting the occurrence of oxidative stress, with increased superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX). Our results showed also a significant upregulation of SOD, GR and APX genes in nodules. Nodule homoglutathione (HGSH) levels increased with the different Ni concentrations, with a remarkable decrease of glutathione S-transferase (GST) activity and glutathione (GSH) content for the highest Ni concentration with 43% and 52% reduction, respectively. The phytochelatin (PC) and metallothionein (MT) concentrations increased in nodules, which implied the triggering of a cellular protection mechanism for coping with Ni toxicity. The results suggested that Ni promotes a drastic oxidative stress in alfalfa nodules, yet the expression of MT and PC to reduce Ni toxicity could be used as Ni stress bioindicators. Our findings provide new insights into the central role of alfalfa nodules in limiting the harmful effects of soil pollution. Therefore, nodules co-expressing antioxidant enzymes may have high phytoremediation potential.