Reactive oxygen species (ROS) are central regulators of plant growth, development, and environmental adaptation, with hydrogen peroxide (H2O2) acting as a key signaling molecule. The ascorbate-glutathione cycle is the major antioxidant pathway that maintains H2O2 homeostasis, and monodehydroascorbate reductase (MDHAR) plays a critical role by regenerating reduced ascorbate (ASC) from monodehydroascorbate (MDHA). Although traditionally viewed as an ASC-recycling enzyme, increasing evidence indicates that MDHAR has broader functions in redox regulation. Recent studies have revealed that MDHAR contributes to stress responses, developmental regulation, and ROS signaling through mechanisms that cannot be fully explained by ASC recycling alone. In this review, we summarize recent advances in the structural characteristics, regulation, subcellular specialization, and evolution of plant MDHARs. We also discuss emerging non-canonical functions of MDHAR, alternative pathways for MDHA reduction, and the mechanistic basis for the contrasting effects of MDHAR manipulation on ASC accumulation. Finally, we propose that MDHAR functions as an integrative hub linking ascorbate metabolism with cellular redox networks and highlight key challenges and future opportunities for exploiting MDHAR to improve crop stress tolerance, productivity, and nutritional quality.

Accumulating evidence shows that reversible protein S-nitrosylation is essential for H2O2 homoeostasis and signalling. However, roles for denitrosylation in such oxidative signalling remain poorly understood. Here, we examined this question using the Arabidopsis catalase-defective mutant, cat2, in which oxidative stress induces both glutathione accumulation and salicylic acid (SA) pathways. Induction of these pathways was accompanied by enhanced thioredoxin (TRXH5) expression, and oxidative stress-induced activation of the SA pathway was compromised when TRXH5 expression was genetically disabled, whereas TRXH5 overexpression stimulates H2O2-triggered SA responses. Intriguingly, TRXh5-reinforced SA responses were antagonised by glutathione (GSH) deficiency when introducing additional pad2 mutation, localised in the GLUTAMATE-CYSTEINE LIGASE gene encoding the first enzyme of glutathione biosynthesis. Further analysis revealed that the two active cysteine residues of recombinant TRXh5 can be denitrosylated by GSH. Blocking glutathione accumulation increased more TRXh5-SNO formation in TRXH5-YFP cat2 pad2 trxh5 than in TRXH5-YFP cat2 trxh5. Furthermore, S-nitrosoglutathione reductase (GSNOR) was capable of physically interacting with TRXh5, and was also required for GSH-dependent TRXh5 denitrosylation and TRXh5-enhanced SA responses during oxidative stress. Collectively, these data suggest that GSH/GSNOR constitutes an active denitrosylating module that works together with the canonical NADPH-dependent TRX-reducing pathway to sustain cytosolic TRXh5 operation within the oxidative signalling framework.

Dear Editor, Spodoptera litura,commonly known as the tobacco cutworm,is a polyphagous agricultural pest worldwide,causing significant economic losses to a wide range of crops.Over the past decades,S.litura has devel-oped high resistance levels to multiple chemical insec-ticides(Li et al.,2024),and shown low susceptibility to transgenic Bacillus thuringiensis(Bt)cotton(Wan et al.,2008).This necessitates the exploration of alternative strategies for effective S.litura control.The RNA in-terference(RNAi)-based approach,employing double-stranded RNA(dsRNA)targeting essential genes in pests,has paved the way for a new generation of insect pest management(Zhu & Palli,2020).However,RNAi effi-ciency in lepidopteran insects has been hindered by the rapid degradation of dsRNA by highly active nucleases in body fluids(Shukla et al.,2016;Guan et al.,2018).

It is well known that high temperatures can lead to a decrease in anthocyanin accumulation in plant organs, but there is insufficient understanding of the internal physiological factors that cause this reduction in anthocyanin accumulation. Apart from the inhibition of synthesis by high temperature, it is not clear how the branch pathways, degradation or transport of anthocyanin change dynamically with temperature increase. Taking red-fleshed kiwifruit as the material, we employed UPLC-MS/MS to investigate the levels of intermediate metabolites in the anthocyanin biosynthesis pathway and potential degradation products. The findings indicated that as the temperature rises, the upstream and downstream branch pathways of the anthocyanin synthesis pathway are adjusted accordingly. Lignin metabolism is enhanced, while the downstream anthocyanin and adjacent branch pathways jointly decrease, but the degradation of anthocyanin accelerated, concurrently with a significant decrease in anthocyanin accumulation. Additionally, the content of a representative degradation product, protocatechuic acid, significantly increased. Kinetic analysis demonstrated that as temperature rose (25-40 °C), the degradation rate constant of anthocyanins increased with the half-life decreased, and the critical temperature range for anthocyanin degradation was between 35 and 40 °C. It is confirmed that under high-temperature conditions, the flow of metabolites in the phenylpropanoid pathway of red-fleshed kiwifruit has undergone readjustment. The inhibition of synthesis and the concurrent degradation jointly contribute to the actual accumulation level of anthocyanins. Through RNA-Seq and enzyme activity experiments, we also identified two genes/enzymes that were promoted by high-temperature stimulation, laccase-12-like (Achn037101) and glucan endo-1,3-β-glucosidase (Achn008121). This study reveals the metabolic kinetics of anthocyanin metabolism in red-fleshed kiwifruit in vivo and offers a deeper understanding of anthocyanin degradation in response to high temperature.

Transposable elements (TEs) are abundant components of plant genomes, yet their transcriptional activity and potential biological roles remain underexplored, especially under environmental stress conditions. This study investigates the transcriptional dynamics of TEs in Brassica napus during drought stress in seed development, aiming to uncover their contributions to stress responses and seed germination. RNA-seq data were analyzed for TE transcriptional activity in wild-type (WT) and BnaABI5 CRISPR-edited mutant lines of B. napus. A comprehensive computational pipeline was used to identify and characterize TE-derived transcripts, including protein-coding and long non-coding RNAs (lncRNAs). Functional annotation was performed for protein-coding TE transcripts located in intergenic regions to predict their involvement in biological processes. Out of 212,800 TEs identified in the B. napus genome, 17,547 were transcriptionally active, yielding 15,808 protein-coding transcripts and 1,739 lncRNAs. Among these, 65 protein-coding TE transcripts were identified as transposase genes, while 860 transcripts were predicted to represent novel genes derived from transposon regions, potentially participating in monocarboxylic acid metabolic processes. Specific to drought stress responses during seed germination, 128 protein-coding TE transcripts (including 5 transposases) and 37 lncRNAs were differentially expressed. Notably, the lncRNA transcripts MSTRG.108925.4 and MSTRG.109003.7 were implicated in regulating the PHD finger protein ALFIN-LIKE 1 (BnA10g0418090), contributing to drought tolerance mechanisms. This study highlights the functional relevance of TE transcription in the context of drought stress during seed germination, providing novel insights into TE-derived genes and lncRNAs as potential regulators of stress responses. These findings expand the understanding of TE biology in plants and offer valuable resources for future efforts to identify drought-resistant genes in B. napus.

RNA interference (RNAi) technology is emerging as a promising and effective alternative for pest control. However, its application has faced challenges due to the inconsistent efficacy of RNAi across different pest species. Factors contributing to this variability include the instability and insufficient internalization of double-stranded RNA (dsRNA). To address these challenges and enhance RNAi efficiency, we have employed bacteriophage MS2 virus-like particles (VLPs) as a delivery platform. MS2 VLPs can autonomously assemble into a capsid and encapsulate RNA molecules with pac site. This approach is utilized to package hpRNA within Escherichia coli for the management of the destructive pest Henosepilachna vigintioctopunctata, a coleopteran insect. We tested three E. coli strains-BL21 (DE3), HT115 (DE3) and BL21 (DE3)-RNase III-for the expression of coat protein (CP) and hpACT. Among these, BL21 (DE3)-RNase III-yielded higher levels of CP and hpACT. Transmission electron microscopy analysis revealed that the CP expressed in BL21 (DE3)-RNase III-successfully encapsulated hpACT within MS2 VLPs. The hpACT encapsulated by MS2 showed resistance to RNase III and the intestinal fluid of H. vigintioctopunctata. By applying BL21 (DE3)-RNase III-cells expressing CP and hpACT through painting or spraying onto the detached leaves of Solanum nigrum and entire plants of S. tuberosum, RNAi efficiency against H. vigintioctopunctata was enhanced. Our study suggests that the potential of the MS2 VLP-coated hpRNA strategy in advancing innovative RNAi-based pest control applications.

Plastid transformation offers valuable benefits in plant biotechnology, such as high-level transgene expression and the absence of gene silencing. Here we describe the first protocol of a plastid transformation system for a woody vine (liana) kiwifruit (Actinidia chinensis). The transgenic DNA carries a spectinomycin-resistance gene (aadA) cassette and a green fluorescent protein (GFP) reporter gene cassette, flanked by two adjacent kiwifruit plastid genome sequences, thereby allowing targeted insertion between the trnfM and trnG genes. Six spectinomycin-resistant shoots were obtained out of 12 plates subjected to bombardment, and two were positive events, confirmed through PCR and Southern blot analyses. The GFP was localized to plastids as monitored by confocal laser scanning microscopy and reached 2.5% of leaf total soluble protein. Success in kiwifruit extends transplastomic technology of woody species beyond poplar, and will provide an attractive biosynthetic chassis for molecular farming.

We report a novel system based on encapsulation of the silencing-inducing RNA in bacteriophage MS2 virus-like particles (VLPs) that efficiently delivers insecticidal RNA molecules to cotton bollworm, the most devastating insect pest of cotton worldwide.

Plastid-mediated RNA interference has emerged as a promising and effective approach for pest management. By expressing high levels of double-stranded RNAs (dsRNAs) in plastid that target essential pest genes, it has been demonstrated to effectively control certain herbivorous beetles and spider mites. However, as plants are sessile organisms, they frequently experience a combination of biotic and abiotic stresses. It remains unclear whether abiotic stress, such as drought stress, influences the accumulation of dsRNAs produced in plastids and its effectiveness in controlling pests. In this study, we aimed to investigate the effects of drought stress on dsACT expression in transplastomic poplar plants and its control efficiency against the willow leaf beetle (Plagiodera versicolora). Our findings revealed that drought stress did not significantly affect the dsRNA contents in transplastomic poplar plants, but it did lead to higher mortality of insect larvae. This increased mortality may be attributed to increased levels of jasmonic acid and cysteine proteinase inhibitor induced by water deficit. These results contribute to understanding of the mechanisms linking water deficit in plants to insect performance and provide valuable insights for implementing appropriate pest control strategies under drought stress conditions.

Magnesium oxide (MgO) porous ceramics with high porosity, compressive strength and low thermal conductivity were prepared by Organic Foam Template Method. The effects of the sintering temperature, polycarboxylic acid (PCE) dispersant and pore size of organic foam template on the properties of MgO porous ceramics were investigated. The experiment results showed that with the increase of sintering temperature, the MgO porous ceramic shrinkage, skeleton density and compressive strength increased. PCE could increase the fluidity of slurry and make the framework clearer, as well as reduce the cracks formed in the process of drying effectively. When the content of PCE was 0.5 wt-%, the porosity, compressive strength and thermal conductivity of MgO porous ceramics were 88.5%, 1.6 MPa and 0.045 W/(m·K), respectively. In addition, as the pore size of the organic foam template decreased, the porosity decreased, and the resistance increased and the thermal conductivity increased.

Cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera Noctuidae) is a major agricultural pest that causes significant yield losses of crop plants each year. In the past few decades, the potent approaches of H. armigera control, including chemical insecticides and Bacillus thuringiensis (Bt) toxins, have been constrained due to health hazards, environmental contamination, and development of resistance, after their extensive application. Thus, there is need to find alternative strategies for H. armigera management. Expression of insect-targeted RNA interference (RNAi) constructs in host plants has emerged as one of such a novel and environment friendly strategies. However, various factors, such as highly active nucleases and high pH in the midgut, as well as failure of small interfering RNA to escape the endosome, lead to relatively low RNAi efficiency in lepidopteran insects. Although most plant-mediated RNAi experiments compromised insect development and growth of H. armigera, full protection of transgenic plants against H. armigera was not achieved and no commercially available product has yet been developed for H. armigera control. A clear understanding of the current status of RNAi utilization in H. armigera control is critical for improving its efficiency. In this review, we summarize the recent advances in plant-mediated RNAi for H. armigera control, including target gene selection, double stranded RNA or artificial microRNA expression from nuclear or plastid genome, and protection of RNAi molecules from degradation by nucleases. Additionally, we highlight factors influencing the efficacy of plant-mediated RNAi, and discuss the future research of this plant genetic engineering strategy for achieving effective management of H. armigera.

Pseudomonas fluorescens group, such as Pseudomonas protegens and Pseudomonas chlororaphis, can be utilized as insect-killing agents. Most insecticidal Pseudomonas described so far have high toxicity for insects of the order Lepidoptera. In this study, Pseudomonas strain PcR3-3 was isolated from the willow root. It showed a high mortality for the coleopteran species Plagiodera versicolora (Coleoptera: Chrysomelidae), but not for the lepidopteran Helicoverpa armigera. Strain PcR3-3 displayed high colonization ability in the P. versicolora compared with P. chlororaphis PCL1391, indicating that the insecticidal activities correlated with the colonization ability of Pseudomonas strain in the host. Phylogenetic analysis of the genome revealed that PcR3-3 belonged to P. chlororaphis subsp. aureofaciens. Numerous insecticidal protein-encoding genes, typical biosynthetic gene clusters for some insecticidal metabolite and type VI secretion system, known to be involved in insect pathogenicity, were present in the P. chlororaphis PcR3-3 genome. However, the insecticidal toxin Fit-encoding gene which commonly presents in P. chlororaphis, was not found in the P. chlororaphis PcR3-3 genome. Furthermore, there are some divergent insecticidal genes between P. chlororaphis PcR3-3 and P. chlororaphis PCL1391. This finding implies that P. chlororaphis PcR3-3 is a promising biocontrol agent for pest management applications. The P. chlororaphis-P. versicolora association can be used as a model system to study the interaction between Pseudomonas and coleopteran insects.

BACKGROUND: Bacillus thuringiensis (Bt) and its crystal toxin or delta-endotoxins (Cry) offer great potential for the efficient control of crop pests. A vast number of pests can potentially infect the same host plant, either simultaneously or sequentially. However, no effective Bt-Cry protein has been reported to control both aphids and plant parasitic nematodes due to its highly specific activity. RESULTS: Our study indicated that the Cry5Ba2 protein was toxic to the green peach aphid Myzus persicae, which had a median lethal concentration (LC50) of 9.7 ng mu L-1 and fiducial limits of 3.1-34.6 ng mu L-1. Immunohistochemical localization of Cry5Ba2 revealed that it could bind to the apical tip of microvilli in midgut regions. Moreover, transgenic tobacco plants expressing Cry5Ba2 exhibited significant resistance to Myzus persicae, as evidenced by reduced insect survival and impaired fecundity, and also intoxicated the Meloidogyne incognita as indicated by a decrease in galls and progeny reproduction. CONCLUSION: In sum, we identified a new aphicidal Bt toxin resource that could simultaneously control both aboveground and belowground pests, thus extending the application range of Bt-based strategy for crop protection. (c) 2024 Society of Chemical Industry.

Cell engineering is one of the core basic courses of biology-related undergraduate majors in ordinary colleges and universities.It has the characteristics of wide interdisciplinary fields and fast content updates.In order to help students better grasp the knowledge points and expand students'knowledge,it is imperative to reform the teaching of cell engineering.This paper takes some chapters in the course of cell engineering as an example to discuss the application of the primary literature guide in teaching,aiming to stimulate students'enthusiasm for learning and cultivate innovative talents for independent learning.

As plants are sessile organisms, they are inevitably exposed to a variety of environmental stimuli that trigger rapid changes in the generation and disposal of reactive oxygen species such as hydrogen peroxide (H2O2). A major H2O2 scavenging system in plant cells is the ascorbate-glutathione cycle, in which ascorbate peroxidase (APX) catalyzes the conversion of H2O2 into water employing ascorbate as specific electron donor. In higher plants, distinct APX isoforms can occur in multiple subcellular compartments, including chloroplasts, mitochondria, and peroxisomes and the cytosol, to modulate organellar and cellular levels of H2O2. It is well established that APX plays crucial roles in protecting plant cells against diverse environmental stresses, as well as in plant growth and development. Apart from ascorbate, recently, APXs have been found to have a broader substrate specificity and possess chaperone activity, hence participating various biological processes. In this review, we describe the antioxidant properties of APXs and highlight their novel roles beyond 'ascorbate peroxidases'.

Double-stranded RNA (dsRNA)-degrading enzyme (dsRNase) is one of the negative factors influencing the RNA interference (RNAi) effect in Colorado potato beetle (CPB). We have previously shown that plastid-mediated RNAi (PM-RNAi) can be utilized to control CPB. In this study, we aimed to test whether silencing dsRNase could increase the RNAi effect in CPB via PM-RNAi. We generated transplastomic potato (Solanum tuberosum) plants expressing dsRNA targeting shibire (dsSHI) of CPB. St-dsSHIplants (transplastomic plants expressing dsSHI) showed much less potent RNAi response than transplastomic potato plants expressing dsRNAs targeting CPB Shrub (St-dsSHR) or beta-Actin (St-dsACT), two CPB-resistant transplastomic lines generated previously. The three types of transplastomic lines have weak (St-dsSHI), moderate (St-dsSHR) and strong (St-dsACT) resistant levels to CPB. Treatment with in vitro-synthesized dsdsRNase1/2 (a fusion dsRNA targeting dsRNase1 and dsRNase2 of CPB) led to enhanced suppression of dsRNase1 and dsRNase2 in CPB larvae fed with St-dsSHIplants and dsRNase2 in CPB larvae fed with St-dsSHR plants. St-dsSHR or St-dsSHIplants showed significantly reduced CPB larva weight or damaged area by CPB feeding. These results demonstrate that dsRNase-silencing approach could improve the effect of PM-RNAi when the silencing of target genes has weak or moderate strength of the insecticidal effects. The findings provide a new proof-of-concept to enhance crop resistance to insect herbivores to via an "RNAi-of-RNAi" approach.

Inducible expression systems can overcome the trade-off between high-level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature-inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature-sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light-dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature-dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pR promoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat-inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost-effective and does not depend on chemical inducers.

RNA interference (RNAi) technology is a promising and effective approach for pest insect management. Owing to its sequence-guided working mechanism, RNAi has a high degree of species-selectivity, thus minimizing potential adverse effects on nontarget organisms. Recently, engineering plastid (chloroplast) genome, rather than the nuclear genome, to produce double-stranded RNAs has emerged as a powerful way to protect plants from multiple arthropod pests. Here, we review the recent progresses in the plastid-mediated RNAi (PM-RNAi) approach for pest control and the factors influencing its efficacy, and propose the strategies for further efficiency improvement. We also discuss the current challenges and the biosafety-related issues of PM-RNAi technology that need to be addressed for commercial production.

The Cas13a-based multiplex RNA targeting system can be engineered to confer resistance to RNA viruses, whereas the number and expression levels of gRNAs have no significant effect on viral interference. The CRISPR–Cas systems provide adaptive immunity to bacterial and archaeal species against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether the number and expression levels of guide RNAs (gRNAs) have effects on the efficiency of RNA virus inhibition is unknown. Here, we repurpose CRISPR/Cas13a in combination with an endogenous tRNA-processing system (polycistronic tRNA–gRNA) to target four genes of potato virus Y (PVY) with varying expression levels. We expressed Cas13a and four different gRNAs in potato lines, and the transgenic plants expressing multiple gRNAs displayed similar suppression of PVY accumulation and reduced disease symptoms as those expressing a single gRNA. Moreover, PTG/Cas13a-transformed plants with different expression levels of multiple gRNAs displayed similar resistance to PVY strains. Collectively, this study suggests that the Cas13a-based multiplex RNA targeting system can be utilized to engineer resistance to RNA viruses in plants, whereas the number and expression levels of gRNAs have no significant effect on CRISPR/Cas13a-mediated viral interference in plants.