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.

APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

Investigation of morpho-physiolgical traits and gene expression in barley under nitrogen deficiency.

Nitrogen (N) is an essential element for plant growth, and its deficiency influences plants at several physiological and gene expression levels. Barley (Hordeum vulgare) is one of the most important food grains from the Poaceae family and one of the most important staple food crops. However, the seed yield is limited by a number of stresses, the most important of which is the insufficient use of N. Thus, there is a need to develop N-use effective cultivars. In this study, comparative physiological and molecular analyses were performed using leaf and root tissues from 10 locally grown barley cultivars. The expression levels of nitrate transporters, HvNRT2 genes, were analyzed in the leaf and root tissues of N-deficient (ND) treatments of barley cultivars after 7 and 14 days following ND treatment as compared to the normal condition. Based on the correlation between the traits, root length (RL) had a positive and highly significant correlation with fresh leaf weight (FLW) and ascorbate peroxidase (APX) concentration in roots, indicating a direct root and leaf relationship with the plant development under ND. From the physiological aspects, ND enhanced carotenoids, chlorophylls a/b (Chla/b), total chlorophyll (TCH), leaf antioxidant enzymes such as ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT), and root antioxidant enzymes (APX and POD) in the Sahra cultivar. The expression levels of HvNRT2.1, HvNRT2.2, and HvNRT2.4 genes were up-regulated under ND conditions. For the morphological traits, ND maintained root dry weight among the cultivars, except for Sahra. Among the studied cultivars, Sahra responded well to ND stress, making it a suitable candidate for barely improvement programs. These findings may help to better understand the mechanism of ND tolerance and thus lead to the development of cultivars with improved nitrogen use efficiency (NUE) in barley.

Genome-wide identification and analysis of ascorbate peroxidase (APX) gene family in hemp (Cannabis sativa L.) under various abiotic stresses.

Ascorbate peroxidase (APX) plays a critical role in molecular mechanisms such as plant development and defense against abiotic stresses. As an important economic crop, hemp (Cannabis sativa L.) is vulnerable to adverse environmental conditions, such as drought, cold, salt, and oxidative stress, which lead to a decline in yield and quality. Although APX genes have been characterized in a variety of plants, members of the APX gene family in hemp have not been completely identified. In this study, we (1) identified eight members of the CsAPX gene family in hemp and mapped their locations on the chromosomes using bioinformatics analysis; (2) examined the physicochemical characteristics of the proteins encoded by these CsAPX gene family members; (3) investigated their intraspecific collinearity, gene structure, conserved domains, conserved motifs, and cis-acting elements; (4) constructed a phylogenetic tree and analyzed interspecific collinearity; and (5) ascertained expression differences in leaf tissue subjected to cold, drought, salt, and oxidative stresses using quantitative real-time-PCR (qRT-PCR). Under all four stresses, CsAPX6, CsAPX7, and CsAPX8 consistently exhibited significant upregulation, whereas CsAPX2 displayed notably higher expression levels under drought stress than under the other stresses. Taken together, the results of this study provide basic genomic information on the expression of the APX gene family and pave the way for studying the role of APX genes in abiotic stress.

Measurements of Ascorbate and Dehydroascorbate in Plants Using High-Performance Liquid Chromatography.

The current concepts emphasize the fundamental role of reactive oxygen species (ROS) as signaling molecules that coordinate defense mechanisms, cell death, and the growth and development processes in plants. However, due to the inherent reactivity of ROS, achieving precise control over their levels within plant cells, both spatially and temporally, becomes important to effectively harness the potential of ROS signaling while concurrently minimizing the risk of oxidative damage. Ascorbate is an exceptional antioxidant and contributes to the antioxidant defense system in plants. Its role is further reinforced by the presence of ascorbate peroxidases and enzymes responsible for recycling ascorbate from its oxidized forms. Ascorbate metabolism plays a pivotal role in averting oxidative damage and facilitates meticulous regulation of ROS signal availability. This chapter outlines the preferred protocol for the measurement of ascorbate.

Detection of Ascorbate Peroxidase (APX) Activity in Plant Tissues: Using Non-denaturing PAGE and Spectrophotometric Assay.

At the cellular level, the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), due to different abiotic or biotic stress, causes oxidative stress that induces an imbalance in the metabolism. Among the different H2O2-scavenging enzymatic antioxidants, ascorbate peroxidase (APX) is a heme-peroxidase that plays an important role in the ascorbate-glutathione pathway using ascorbate to reduce H2O2 to water. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a spectrophotometric assay for APX activity, the protocol allows identifying diverse APX isozymes present in different organs and plant species.

Correlating Structure with Spectroscopy in Ascorbate Peroxidase Compound II.

Structural and spectroscopic investigations of compound II in ascorbate peroxidase (APX) have yielded conflicting conclusions regarding the protonation state of the crucial Fe(IV) intermediate. Neutron diffraction and crystallographic data support an iron(IV)-hydroxo formulation, whereas Mössbauer, X-ray absorption (XAS), and nuclear resonance vibrational spectroscopy (NRVS) studies appear consistent with an iron(IV)-oxo species. Here we examine APX with spectroscopy-oriented QM/MM calculations and extensive exploration of the conformational space for both possible formulations of compound II. We establish that irrespective of variations in the orientation of a vicinal arginine residue and potential reorganization of proximal water molecules and hydrogen bonding, the Fe-O distances for the oxo and hydroxo forms consistently fall within distinct, narrow, and nonoverlapping ranges. The accuracy of geometric parameters is validated by coupled-cluster calculations with the domain-based local pair natural orbital approach, DLPNO-CCSD(T). QM/MM calculations of spectroscopic properties are conducted for all structural variants, encompassing Mössbauer, optical, X-ray absorption, and X-ray emission spectroscopies and NRVS. All spectroscopic observations can be assigned uniquely to an Fe(IV)═O form. A terminal hydroxy group cannot be reconciled with the spectroscopic data. Under no conditions can the Fe(IV)═O distance be sufficiently elongated to approach the crystallographically reported Fe-O distance. The latter is consistent only with a hydroxo species, either Fe(IV) or Fe(III). Our findings strongly support the Fe(IV)═O formulation of APX-II and highlight unresolved discrepancies in the nature of samples used across different experimental studies.

Ascorbate peroxidase in fruits and modulation of its activity by reactive species.

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate-glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source. APX is present in all photosynthetic eukaryotes from algae to higher plants and, at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including the apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. APX activity can be modulated by various post-translational modifications including tyrosine nitration, S-nitrosation, persulfidation, and S-sulfenylation. This allows the connection of H2O2 metabolism with other relevant signaling molecules such as NO and H2S, thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process and during post-harvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and, more recently, melatonin is seen as a new alternative to maintain and extend the shelf life and quality of fruits because they can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered as new biotechnological tools to improve crop quality in the horticultural industry.

Physiological function and regulation of ascorbate peroxidase isoforms.

Ascorbate peroxidase (APX) reduces H2O2 to H2O by utilizing ascorbate as a specific electron donor and constitutes the ascorbate-glutathione cycle in organelles of plants including chloroplasts, cytosol, mitochondria, and peroxisomes. It has been almost 40 years since APX was discovered as an important plant-specific H2O2-scavenging enzyme, during which time many research groups have conducted molecular physiological analyses. It is now clear that APX isoforms function not only just as antioxidant enzymes but also as important factors in intracellular redox regulation through the metabolism of reactive oxygen species. The function of APX isoforms is regulated at multiple steps, from the transcriptional level to post-translational modifications of enzymes, thereby allowing them to respond flexibly to ever-changing environmental factors and physiological phenomena such as cell growth and signal transduction. In this review, we summarize the physiological functions and regulation mechanisms of expression of each APX isoform.

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.

Antioxidative response of parthenocarpic tomato, iaa9-3 and iaa9-5, under heat stress condition.

It has previously been shown that parthenocarpic tomato mutants, iaa9-3 and iaa9-5, can adapt, grow, and produce fruit under heat-stress conditions. However, the physiological processes in those two mutants especially for the enzymatic system that works to neutralize ROS are not clear. The objective of this research was to determine how the scavenging enzyme system responds to the heat stress in those mutants. The iaa9-3, iaa9-5, and WT-MT as a control were cultivated under two environmental conditions; normal and heat stress conditions. Vegetative and reproductive growth were observed during cultivation period. The activities of catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) were investigated in both wild-type and parthenocarpic tomato mutants under normal and heat stress conditions. The results showed that under heat stress condition, the mutants, iaa9-3 and iaa9-5, and WT-MT resulted in reduction of the vegetative growth, but those mutants showed better growth than WT-MT. Higher chlorophyll content in iaa9-3 and iaa9-5 was observed under normal or heat stress condition. Despite their growth reduction under heat stress conditions, iaa9-3 and iaa9-5 resulted in the significant higher CAT, APX and SOD activity than WT-MT. The results suggest that higher chlorophyll content and enhanced CAT, APX and SOD activity in the iaa9-3 and iaa9-5 mutants are adaptive strategies to survive in heat stress conditions.

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.

Cold priming on pathogen susceptibility in the Arabidopsis eds1 mutant background requires a functional stromal Ascorbate Peroxidase.

24 h cold exposure (4°C) is sufficient to reduce pathogen susceptibility in Arabidopsis thaliana against the virulent Pseudomonas syringae pv. tomato (Pst) strain even when the infection occurs five days later. This priming effect is independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) and can be observed in the immune-compromised eds1-2 null mutant. In contrast, cold priming-reduced Pst susceptibility is strongly impaired in knock-out lines of the stromal and thylakoid ascorbate peroxidases (sAPX/tAPX) highlighting their relevance for abiotic stress-related increased immune resilience. Here, we extended our analysis by generating an eds1 sapx double mutant. eds1 sapx showed eds1-like resistance and susceptibility phenotypes against Pst strains containing the effectors avrRPM1 and avrRPS4. In comparison to eds1-2, susceptibility against the wildtype Pst strain was constitutively enhanced in eds1 sapx. Although a prior cold priming exposure resulted in reduced Pst titers in eds1-2, it did not alter Pst resistance in eds1 sapx. This demonstrates that the genetic sAPX requirement for cold priming of basal plant immunity applies also to an eds1 null mutant background.

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.

The effect of foliar application of Ascophyllum nodosum (L.) Le Jol. seaweed extract on biochemical traits related to abiotic stresses in pistachio (Pistacia vera L. cv. Kaleh-Ghoochi).

BACKGROUND: Due to the important economic role of pistachio (Pistacia vera L.) the cultivation of this valuable crop has been extended. Various abiotic stresses harm the growth and performance of pistachio. Seaweed extract containing various substances such as pseudo-hormones that stimulate growth, nutritional elements, and anti-stress substances can cause more resistance to abiotic stresses, and increase the quantity and the quality of the fruit. The present study was conducted to evaluate the effect of foliar application of Ascophyllum nodosum (L.) Le Jol. seaweed extract on some biochemical traits related to abiotic stress in Pistacia vera L. cv. Kaleh-Ghoochi. The first factor of foliar spraying treatment included A. nodosum seaweed extract at four levels (0, 1, 2, and 3 g/L), and the second factor was the time of spraying solution which was done at three times (1- at the beginning of pistachio kernel growth period at the end of June, 2- at the stage of full kernel development at the end of August, and 3- Spraying in both late June and August). RESULTS: The results showed that all investigated traits were significant under the treatment of seaweed extract compared with the control. The seaweed extract concentrations had a significant effect on all traits except soluble carbohydrates, but the time of consumption of seaweed extract on soluble carbohydrates, protein, peroxidase, ascorbate peroxidase, and superoxide dismutase enzymes was significant, while had no significant effect on the rest of the traits. According to the interaction effect of time and concentration of consumption of seaweed extract, the highest values of the biochemical characters were as follows: total phenol content: 168.30 mg CAE/g DW, flavonoid content: mg CE/g DW, catalase: 12.66 µmol APX min- 1 mg- 1 protein, superoxide dismutase: 231.4 µmol APX min- 1 mg- 1 protein, and ascorbate peroxidase: 39.53 µmol APX min- 1 mg- 1 protein. CONCLUSIONS: Based on the results of this study, it seems that it is possible to use fertilizers containing A. nodosum seaweed extract with a concentration of 3 g/L in August to increase the tolerance of the pistachio cultivar "Kaleh-Ghoochi" to abiotic stresses.

Genome-Wide Identification, Evolutionary Analysis, and Functional Studies of APX Genes in Melon (Cucuis melo L.).

The antioxidative enzyme ascorbate peroxidase (APX) exerts a critically important function through scavenging reactive oxygen species (ROS), alleviating oxidative damage in plants, and enhancing their tolerance to salinity. Here, we identified 28 CmAPX genes that display an uneven distribution pattern throughout the 12 chromosomes of the melon genome by carrying out a bioinformatics analysis. Phylogenetic analyses revealed that the CmAPX gene family comprised seven different clades, with each clade of genes exhibiting comparable motifs and structures. We cloned 28 CmAPX genes to infer their encoded protein sequences; we then compared these sequences with proteins encoded by rice APX proteins (OsAPX2), Puccinellia tenuiflora APX proteins (PutAPX) and with pea APX proteins. We found that the CmAPX17, CmAPX24, and CmAPX27 genes in Clade I were closely related, and their structures were highly conserved. CmAPX27 (MELO3C020719.2.1) was found to promote resistance to 150 mM NaCl salt stress, according to quantitative real-time fluorescence PCR. Transcriptome data revealed that CmAPX27 was differentially expressed among tissues, and the observed differences in expression were significant. Virus-induced gene silencing of CmAPX27 significantly decreased salinity tolerance, and CmAPX27 exhibited differential expression in the leaf, stem, and root tissues of melon plants. This finding demonstrates that CmAPX27 exerts a key function in melon's tolerance to salt stress. Generally, CmAPX27 could be a target in molecular breeding efforts aimed at improving the salt tolerance of melon; further studies of CmAPX27 could unveil novel physiological mechanisms through which antioxidant enzymes mitigate the deleterious effects of ROS stress.

High-Proportion Blue Light Irradiation at the End-of-Production Stage Promotes the Biosynthesis and Recycling of Ascorbate in Lettuce.

Ascorbate (AsA), an essential antioxidant for both plants and the human body, plays a vital role in maintaining proper functionality. Light plays an important role in metabolism of AsA in horticultural plants. Our previous research has revealed that subjecting lettuce to high light irradiation (HLI) (500 µmol·m-2·s-1) at the end-of-production (EOP) stage effectively enhances AsA levels, while the optimal light quality for AsA accumulation is still unknown. In this study, four combinations of red (R) and blue (B) light spectra with the ratio of 1:1 (1R1B), 2:1 (2R1B), 3:1 (3R1B), and 4:1 (4R1B) were applied to investigate the biosynthesis and recycling of AsA in lettuce. The results demonstrated that the AsA/total-AsA content in lettuce leaves was notably augmented upon exposure to 1R1B and 2R1B. Interestingly, AsA levels across all treatments increased rapidly at the early stage (2-8 h) of irradiation, while they increased slowly at the late stage (8-16 h). The activity of L-galactono-1,4-lactone dehydrogenase was augmented under 1R1B treatment, which is pivotal to AsA production. Additionally, the activities of enzymes key to AsA cycling were enhanced by 1R1B and 2R1B treatments, including ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase. Notably, hydrogen peroxide and malondialdehyde accumulation increased dramatically following 16 h of 1R1B and 2R1B treatments. In addition, although soluble sugar and starch contents were enhanced by EOP-HLI, this effect was comparatively subdued under the 1R1B treatment. Overall, these results indicated that AsA accumulation was improved by irradiation with a blue light proportion of over 50% in lettuce, aligning with the heightened activities of key enzymes responsible for AsA synthesis, as well as the accrual of hydrogen peroxide. The effective strategy holds the potential to enhance the nutritional quality of lettuce while bolstering its antioxidant defenses.

Impairment in photosynthesis induced by CAT inhibition depends on the intensity of photorespiration and peroxisomal APX expression in rice.

We have previously shown that rice plants silenced for peroxisomal ascorbate peroxidase (OsAPX4-RNAi) display higher resilience to photosynthesis under oxidative stress and photorespiratory conditions. However, the redox mechanisms underlying that intriguing response remain unknown. Here, we tested the hypothesis that favorable effects triggered by peroxisomal APX deficiency on photosynthesis resilience under CAT inhibition are dependent on the intensity of photorespiration associated with the abundance of photosynthetic and redox proteins. Non-transformed (NT) and OsAPX4-RNAi silenced rice plants were grown under ambient (AC) or high CO2 (HC) conditions and subjected to 3-amino-1,2,4-triazole (3-AT)-mediated CAT activity inhibition. Photosynthetic measurements evidenced that OsAPX4-RNAi plants simultaneously exposed to CAT inhibition and HC lost the previously acquired advantage in photosynthesis resilience displayed under AC. Silenced plants exposed to environment photorespiration and CAT inhibition presented lower photorespiration as indicated by smaller Gly/Ser and Jo/Jc ratios and glycolate oxidase activity. Interestingly, when these silenced plants were exposed to HC and CAT-inhibition, they exhibited an inverse response compared to AC in terms of photorespiration indicators, associated with higher accumulation of proteins. Multivariate and correlation network analyses suggest that the proteomics changes induced by HC combined with CAT inhibition are substantially different between NT and OsAPX4-RNAi plants. Our results suggest that the intensity of photorespiration and peroxisomal APX-mediated redox signaling are tightly regulated under CAT inhibition induced oxidative stress, which can modulate the photosynthetic efficiency, possibly via a coordinated regulation of protein abundance and rearrangement, ultimately triggered by crosstalk involving H2O2 levels related to CAT and APX activities in peroxisomes.