Evaluation of oxidative stress, biochemical parameters and in silico markers in different pea accessions in response to drought stress.

KEY MESSAGE: ARG6 and ARG10 pea accessions exhibited better tolerance to drought by keeping drought-associated attributes stable and higher, that is, stable chlorophyll content, high antioxidant activity, and the presence of polymorphic bands with stress-responsive EST-SSR markers. Each year, a significant portion of crops is lost due to various abiotic stresses, and even pea (Pisum sativum) crop growth and yield are severely affected by the challenges posed by drought stress. Drought is a critical factor that limits crop growth and development, and its impact is exacerbated by changes in the magnitude of climatic conditions. Drought induces oxidative stress in plants, leading to the accumulation of high concentrations of reactive oxygen species that damage cell structures and vital functioning of cells. The primary objective was to identify stress-tolerant plants by evaluating different morphological and biochemical attributes, such as biomass, chlorophyll content, relative water content, ascorbate peroxidase (APX), superoxide dismutase (SOD), and DPPH scavenging activity, as well as protein, proline, and phenolic content. Our study revealed that pea accessions (ARG6 and ARG10) were more resilient to drought stress as their chlorophyll, relative water, protein, and proline contents increased under drought conditions. Antioxidant enzymes, such as SOD, APX, and DPPH activities, also increased under drought stress in ARG10 and ARG6, suggesting that these accessions could bolster the antioxidant defense system in response to drought stress. Based on putative (cellular, biological, and metabolic) functions, ten EST-SSR primers were selected for the amplification study. Three EST-SSR primers, AUMP06_110, AUMP18_300, and AUMP31_250, were used for ARG6 and ARG10. Based on the correlation between the presence or absence of specific EST-SSR alleles, various physiological and morphological traits, and DPPH scavenging activity, both ARG10 and ARG6 demonstrated resistance to drought stress.

Alternative oxidase affects ascorbate metabolism in Arabidopsis thaliana plants under high-light conditions: possible links between respiratory electron transport pathways in mitochondria.

Some aspects of the relationship between ascorbate (Asc) metabolism and the functioning of mitochondrial alternative oxidase (AOX) under moderately high light (MHL, 400 µmol m-2 s-1) using Arabidopsis thaliana mutant lines were studied. After 8 h of MHL in the AOX1a antisense line (AS-12), decreasing the relative reduced Asc pool due to increased ascorbate peroxidase activity was accompanied by the accumulation of a pool of the other highly effective antioxidant - glutathione. In the vitamin C-deficient line (vtc2), VTC2 expression and the Asc pool were expectedly low, and after 8 h of MHL, dehydroascorbate (DHA) content was increased, although slight activation of AOX and L-galacton-1,4-lactone dehydrogenase was detected. In the AOX-inhibited vtc2 line after 8 h of MHL, both the DHA levels and cytochrome pathway capacity increased. Interestingly, the suppression of AOX in the AS-12 line had a negative effect on the ATP content, whereas the activation of AOX in the vtc2 line improved the energy balance in MHL conditions. Possible mechanisms of interaction at the level of the ascorbate-glutathione hub and mitochondrial electron transport pathways, mediated through Asc synthesis, for the regulation of energy balance and Asc metabolism during plant adaptation to high light are discussed.

A truncated B-box zinc finger transcription factor confers drought sensitivity in modern cultivated tomatoes.

Enhancing drought tolerance in crops and understanding the underlying mechanisms have been subject of intense research. The precise function and molecular mechanisms of B-box zinc finger proteins (BBX) remain elusive. Here, we report a natural allele of BBX18 (BBX18TT) that encodes a C-terminal truncated protein. While most wild tomato germplasms contain the BBX18CC allele and show more drought tolerant, modern cultivated tomatoes mostly carry BBX18TT allele and are more drought sensitive. Knockout of BBX18 leads to improved drought tolerance in transgenic plants of cultivated tomato. Ascorbate peroxidase 1 (APX1) is identified as a BBX18-interacting protein that acts as a positive regulator of drought resistance in tomato. Chromatin immunoprecipitation sequencing analyses reveal that BBX18 binds to a unique cis-acting element of the APX1 promoter and represses its gene expression. This study provides insights into the molecular mechanism underlying drought resistance mediated by the BBX18-APX1 module in plants.

Promoter role of putrescine for molecular and biochemical processes under drought stress in barley.

Drought, which adversely affects plant growth and continuity of life and reduces product yield and quality, is one of the most common abiotic stresses at the globally. One of the polyamines that regulates plant development and reacts to abiotic stressors, including drought stress, is Putrescine (Put). This study compared the physiological and molecular effects of applying exogenous Put (10 µM) to barley (Hordeum vulgare cv. Burakbey) under drought stress (- 6.30 mPa PEG 6000). The 21-day drought stress imposed on the barley plant had a strong negative effect on plant metabolism in all experimental groups. Exogenous Put treatment under drought stress had a reformative effect on the cell cycle (transitions from G0-G1 to S and from S to G2-M), total protein content (almost 100%), endogenous polyamine content, malondialdehyde (MDA) (70%), and ascorbate peroxidase (APX) (62%) levels compared to the drought stress plants. Superoxide dismutase (SOD) (12%) and catalase (CAT) (32%) enzyme levels in the same group increased further after exogenous Put application, forming a response to drought stress. Consequently, it was discovered that the administration of exogenous Put in barley raises endogenous polyamine levels and then improves drought tolerance due to increased antioxidant capability, cell division stimulation, and total protein content.

Genome-Wide Identification of APX Gene Family in Citrus maxima and Expression Analysis at Different Postharvest Preservation Times.

Ascorbate peroxidase (APX) is a crucial enzyme involved in cellular antioxidant defense and plays a pivotal role in modulating reactive oxygen species (ROS) levels under various environmental stresses in plants. This study utilized bioinformatics methods to identify and analyze the APX gene family of pomelo, while quantitative real-time PCR (qRT-PCR) was employed to validate and analyze the expression of CmAPXs at different stages of fruit postharvest. This study identified 96 members of the CmAPX family in the entire pomelo genome, with uneven distribution across nine chromosomes and occurrences of gene fragment replication. The subcellular localization includes peroxisome, cytoplasm, chloroplasts, and mitochondria. The CmAPX family exhibits a similar gene structure, predominantly consisting of two exons. An analysis of the upstream promoter regions revealed a significant presence of cis-acting elements associated with light (Box 4, G-Box), hormones (ABRE, TCA-element), and stress-related (MBS, LTR, ARE) responses. Phylogenetic and collinearity analyses revealed that the CmAPX gene family can be classified into three subclasses, with seven collinear gene pairs. Furthermore, CmAPXs are closely related to citrus, pomelo, and lemon, followed by Arabidopsis, and exhibit low homology with rice. Additionally, the transcriptomic heat map and qPCR results revealed that the expression levels of CmAPX57, CmAPX34, CmAPX50, CmAPX4, CmAPX5, and CmAPX81 were positively correlated with granulation degree, indicating the activation of the endogenous stress resistance system in pomelo cells by these genes, thereby conferring resistance to ROS. This finding is consistent with the results of GO enrichment analysis. Furthermore, 38 miRNAs were identified as potential regulators targeting the CmAPX family for post-transcriptional regulation. Thus, this study has preliminarily characterized members of the APX gene family in pomelo and provided valuable insights for further research on their antioxidant function and molecular mechanism.

APEX2-Mediated Proximity Protein Labeling in Dictyostelium.

Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

Ascorbate peroxidase catalyses synthesis of protocatechualdehyde from p-hydroxybenzaldehyde in Lycoris aurea.

Protocatechualdehyde is a plant natural phenolic aldehyde and an active ingredient with important bioactivities in traditional Chinese medicine. Protocatechualdehyde is also a key intermediate in the synthesis of Amaryllidaceae alkaloids for supplying the C6-C1 skeleton. However, the biosynthesis of protocatechualdehyde in plants remains obscure. In this study, we measured the protocatechualdehyde contents in the root, bulb, scape and flower of the Amaryllidaceae plant Lycoris aurea (L'Hér.) Herb., and performed the correlation analysis between the protocatechualdehyde contents and the transcriptional levels of the phenolic oxidization candidate protein encoding genes. We found that a novel ascorbate peroxidase encoded by the contig_24999 in the L. aurea transcriptome database had potential role in the biosynthesis of protocatechualdehyde. The LauAPX_24999 gene was then cloned from the cDNA of the scape of L. aurea. The transient expression of LauAPX_24999 protein in Arabidopsis protoplasts demonstrated that LauAPX_24999 protein was localized in the cytoplasm, thus belonging to Class II L-ascorbate peroxidase. Subsequently, LauAPX_24999 protein was heterogenously expressed in Escherichia coli, and identified that LauAPX_24999 biosynthesized protocatechualdehyde from p-hydroxybenzaldehyde using L-ascorbic acid as the electron donor. The protein structure modelling and molecular docking indicated that p-hydroxybenzaldehyde could access to the active pocket of LauAPX_24999 protein, and reside at the δ-edge of the heme group while L-ascorbic acid binds at the γ-heme edge. To our knowledge, LauAPX_24999 is the first enzyme discovered in plants able to biosynthesize protocatechualdehyde from p-hydroxybenzaldehyde, and offers a competent enzyme resource for the biosynthesis of Amaryllidaceae alkaloids via synthetic biology.

The transcription factor MhZAT10 enhances antioxidant capacity by directly activating the antioxidant genes MhMSD1, MhAPX3a and MhCAT1 in apple rootstock SH6 (Malus honanensis × M. domestica).

Stress tolerance in apple (Malus domestica) can be improved by grafting to a stress-tolerant rootstock, such as 'SH6' (Malus honanensis × M. domestica 'Ralls Genet'). However, the mechanisms of stress tolerance in this rootstock are unclear. In Arabidopsis (Arabidopsis thaliana), the transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 10 is a key component of plant tolerance to multiple abiotic stresses and positively regulates antioxidant enzymes. However, how reactive oxygen species are eliminated upon activation of ZINC FINGER OF ARABIDOPSIS THALIANA 10 in response to abiotic stress remains elusive. Here, we report that MhZAT10 in the rootstock SH6 directly activates the transcription of three genes encoding the antioxidant enzymes MANGANESE SUPEROXIDE DISMUTASE 1 (MhMSD1), ASCORBATE PEROXIDASE 3A (MhAPX3a) and CATALASE 1 (MhCAT1) by binding to their promoters. Heterologous expression in Arabidopsis protoplasts showed that MhMSD1, MhAPX3a and MhCAT1 localize in multiple subcellular compartments. Overexpressing MhMSD1, MhAPX3a or MhCAT1 in SH6 fruit calli resulted in higher superoxide dismutase, ascorbate peroxidase and catalase enzyme activities in their respective overexpressing calli than in those overexpressing MhZAT10. Notably, the calli overexpressing MhZAT10 exhibited better growth and lower reactive oxygen species levels under simulated osmotic stress. Apple SH6 plants overexpressing MhZAT10 in their roots via Agrobacterium rhizogenes-mediated transformation also showed enhanced tolerance to osmotic stress, with higher leaf photosynthetic capacity, relative water content in roots and antioxidant enzyme activity, as well as less reactive oxygen species accumulation. Overall, our study demonstrates that the transcription factor MhZAT10 synergistically regulates the transcription of multiple antioxidant-related genes and elevates reactive oxygen species detoxification.

Knockout of stigmatic ascorbate peroxidase 1 (APX1) delays pollen rehydration and germination by mediating ROS homeostasis in Brassica napus L.

The successful interaction between pollen and stigma is a critical process for plant sexual reproduction, involving a series of intricate molecular and physiological events. After self-compatible pollination, a significant reduction in reactive oxygen species (ROS) production has been observed in stigmas, which is essential for pollen grain rehydration and subsequent pollen tube growth. Several scavenging enzymes tightly regulate ROS homeostasis. However, the potential role of these ROS-scavenging enzymes in the pollen-stigma interaction in Brassica napus remains unclear. Here, we showed that the activity of ascorbate peroxidase (APX), an enzyme that plays a crucial role in the detoxification of hydrogen peroxide (H2O2), was modulated depending on the compatibility of pollination in B. napus. We then identified stigma-expressed APX1s and generated pentuple mutants of APX1s using CRISPR/Cas9 technology. After compatible pollination, the BnaAPX1 pentuple mutants accumulated higher levels of H2O2 in the stigma, while the overexpression of BnaA09.APX1 resulted in lower levels of H2O2. Furthermore, the knockout of BnaAPX1 delayed the compatible response-mediated pollen rehydration and germination, which was consistent with the effects of a specific APX inhibitor, ρ-Aminophenol, on compatible pollination. In contrast, the overexpression of BnaA09.APX1 accelerated pollen rehydration and germination after both compatible and incompatible pollinations. However, delaying and promoting pollen rehydration and germination did not affect the seed set after compatible and incompatible pollination in APX1 pentuple mutants and overexpression lines, respectively. Our results demonstrate the fundamental role of BnaAPX1 in pollen rehydration and germination by regulating ROS homeostasis during the pollen-stigma interaction in B. napus.

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.

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.

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.

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.

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.

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.

Multivariate analysis compares and evaluates drought and flooding tolerances of maize germplasm.

Drought and flooding are the two most important environmental factors limiting maize (Zea mays L.) production globally. This study aimed to investigate the physiological mechanisms and accurate evaluation indicators and methods of maize germplasm involved in drought and flooding stresses. The twice replicated pot experiments with 60 varieties, combined with the field validation experiment with 3 varieties, were conducted under well-watered, drought, and flooding conditions. Most varieties exhibited stronger tolerance to drought than flooding due to higher antioxidant enzyme activities, osmotic adjustment substances, and lower reactive oxygen species. In contrast, flooding stress resulted in higher levels of reactive oxygen species (particularly O2-), ascorbate peroxidase, catalase, peroxidase, and soluble sugars but lower levels of superoxide dismutase, proline, and soluble protein compared with well-watered conditions. Superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, proline, soluble sugars, and protein contents, in addition to plant height, leaf area/plant, and stem diameter, were accurate and representative indicators for evaluating maize tolerance to drought and flooding stresses and could determine a relatively high mean forecast accuracy of 100.0% for the comprehensive evaluation value. A total of 4 principal components were extracted, in which different principal components played a vital role in resisting different water stresses. Finally, the accuracy of the 3 varieties screened by multivariate analysis was verified in the field. This study provides insights into the different physiological mechanisms and accurate evaluation methods of maize germplasm involved in drought and flooding stresses, which could be valuable for further research and breeding.

Identification of citrus APX gene family and their response to CYVCV infection.

Ascorbate peroxidase (APX) is one of the most important antioxidant enzymes in the reactive oxygen metabolic pathway of plants. The role of APX under biotic and abiotic stress conditions has been explored, but the response pattern of APX under biotic stresses is relatively less known. In this study, seven CsAPXs gene family members were identified based on the sweet orange (Citrus sinensis) genome and subjected to evolutionary and structural analysis using bioinformatics software. The APX genes of lemon (ClAPXs) were cloned and showed a high conservation to CsAPXs by sequences alignment. In citrus yellow vein clearing virus (CYVCV)-infected Eureka lemons (C. limon) at 30th day post inoculation, APX activity and accumulation of hydrogen peroxide (H2O2) and malondialdehyde were measured to be 3.63, 2.29, and 1.73 times to that of the healthy control. The expression levels of 7 ClAPX genes in different periods of CYVCV-infected Eureka lemon were analyzed. Notably, ClAPX1, ClAPX5, and ClAPX7 showed higher expression levels compared to healthy plants, while ClAPX2, ClAPX3, and ClAPX4 showed lower expression levels. Functional identification of ClAPX1 in Nicotiana benthamiana showed that increasing the expression of ClAPX1 could significantly reduce the accumulation of H2O2, and it was verified that ClAPX1 is located in the plasma membrane of the cell. The present study provided information on the evolution and function of citrus APXs and revealed for the first time their response pattern to CYVCV infection.

Induced defense responses in cultivated and wild chickpea genotypes against Helicoverpa armigera infestation.

Five desi (GL 12,021, GL 29,095, GL 29,078, H11 22 and CSJ 515) and three wild (GLW 22, GLW 58 and GLW 187) chickpea cultivars showed induced defense response against Helicoverpa armigera infestation as a result of enhanced activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione reductase, polyphenol oxidase, phenylalanine ammonia lyase, tyrosine ammonia lyase in leaves, pod walls and seeds. Catalase activity increased in leaves of GL 12,021, H11 22, GL 29,095, CSJ 515, GLW 22, and GL 29,078 after infestation compared to resistant check; catalase and peroxidase activities in GL 29,095 and GL 29,078; ascorbate peroxidase and glutathione reductase activities in leaves of GLW 58. The increased activity of superoxide dismutase in pod wall of H1122; catalase in pod wall of 29,078, GL 29,095 and GL 22; ascorbate peroxidase and glutathione reductase in pod wall of GLW 58; phenylalanine ammonia lyase and tyrosine ammonia lyase in pod wall of GLW 187, H11 22, GL 20,978, GLW 22 and GLW 58 after infestation as compared to resistant check might be responsible for mitigating infestation induced oxidative stress. MDA content decreased in leaves, pod wall and seeds of GLW 187 and GL 12,021 after infestation. Lower percent pod damage (9.58-12.44%) in GL 12,021, GLW 187, GL 29,095, H11 22, GL 29,078, GLW 22 and GLW 58 as compared to resistant (16.18%) and susceptible (21.50) checks might be attributed to differential induced defense mechanism in them. The identified desi and wild genotypes might be used in breeding program to develop cultivars with improved resistance to herbivore.