Search Results (65)

Alternative oxidase (AOX) serves as a critical terminal oxidase within the plant respiratory pathway, playing a significant role in cellular responses to various stresses. Foxtail millet (Setaria italica), a crop extensively cultivated across Asia, is renowned for its remarkable tolerance to abiotic stresses and minimal requirement for fertilizer. In this study, we conducted a comprehensive genome-wide identification of AOX genes in foxtail millet genome, discovering a total of five SiAOX genes. Phylogenetic analysis categorized these SiAOX members into two subgroups. Prediction of cis-elements within the promoter regions, coupled with co-expression network analysis, intimated that SiAOX proteins are likely involved in the plant’s adaptive response to abiotic stresses. Employing RNA sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR), we scrutinized the expression patterns of the SiAOX genes across a variety of tissues and under multiple abiotic stress conditions. Specifically, our analysis uncovered that SiAOX1, SiAOX2, SiAOX4, and SiAOX5 display distinct tissue-specific expression profiles. Furthermore, SiAOX2, SiAOX3, SiAOX4, and SiAOX5 exhibit responsive expression patterns under abiotic stress conditions, with significant differences in expression levels observed between the shoot and root tissues of foxtail millet seedlings. Haplotype analysis of SiAOX4 and SiAOX5 revealed that these genes are in linkage disequilibrium, with Hap_2 being the superior haplotype for both, potentially conferring enhanced cold stress tolerance in the cultivar group. These findings suggest that both SiAOX4 and SiAOX5 may be targeted for selection in future breeding programs aimed at improving foxtail millet’s resilience to cold stress. Full article

Crassostrea hongkongensis (C. hongkongensis) is one of the three most commonly cultivated oyster species in China. Seasonal hypoxia is one of the most serious threats to its metabolism, reproductive behavior, and survival. To investigate the effects of hypoxia stress on the antioxidant capacity and energy metabolism of C. hongkongensis, the total antioxidant capacity (T-AOC), glycogen content, and enzyme activities (phosphofructokinase, PFK; pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK) of oysters were determined under normoxic (DO 6 ± 0.2 mg/L) and hypoxic (DO 1.5 mg/L) conditions at 0 h, 6 h, 48 h, and 72 h. We also determined the T-AOC, glycogen content, and enzyme activities of oysters under reoxygenation (recovered to normoxia for 24 h). To further examine the potential molecular regulatory mechanism of hypoxic adaptation, a transcriptome analysis was conducted on the gill of C. hongkongensis under normoxia (N, 72 h), hypoxia (H, 72 h), and reoxygenation (R). After being exposed to hypoxia for 6 h, the T-AOC, glycogen content, and enzyme activities of PK, PFK, and PEPCK in C. hongkongensis were significantly decreased. However, after prolonging the duration of hypoxia exposure for 72 h, the T-AOC, glycogen content, and enzyme activities increased compared to that of 48 h. After 24 h reoxygenation, the T-AOC, glycogen content, and enzyme activity of PK and PFK returned to close to initial levels. In addition, a transcriptome analysis discovered 6097 novel genes by mapping the C. hongkongensis genome with the clean reads. In total, 352 differentially expressed genes (DEGs) were identified in the H vs. N comparison group (235 upregulated and 117 downregulated genes). After recovery to normoxia, 292 DEGs (134 upregulated and 158 downregulated genes) were identified in the R vs. N comparison group, and 632 DEGs were identified (253 upregulated and 379 downregulated genes) in the R vs. H comparison group. The DEGs included some hypoxia-tolerant genes, such as phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial (AOX), tyramine beta-hydroxylase (TBH), superoxide dismutase (SOD), glutathione S-transferase (GST), and egl nine homolog 1 isoform X2 (EGLN1). Additionally, DEGs were significantly enriched in the KEGG pathways that are involved in hypoxia tolerance, including the metabolism of xenobiotics by cytochrome P450 pathways and the HIF-1 signaling pathway. Then, we selected the five hypoxic-tolerant candidate DEGs for real-time quantitative polymerase chain reaction (RT-qPCR) validation, and the results were consistent with the transcriptome sequencing data. These discoveries have increased our understanding of hypoxia tolerance, recovery ability after reoxygenation, and molecular mechanisms governing the responses to hypoxia in C. hongkongensis. Full article

The alternative oxidase (AOX), a common terminal oxidase in the electron transfer chain (ETC) of plants, plays a crucial role in stress resilience and plant growth and development. Oat (Avena sativa), an important crop with high nutritional value, has not been comprehensively studied regarding the AsAOX gene family. Therefore, this study explored the responses and potential functions of the AsAOX gene family to various abiotic stresses and their potential evolutionary pathways. Additionally, we conducted a genome-wide analysis to explore the evolutionary conservation and divergence of AOX gene families among three Avena species (Avena sativa, Avena insularis, Avena longiglumis) and four Poaceae species (Avena sativa, Oryza sativa, Triticum aestivum, and Brachypodium distachyon). We identified 12 AsAOX, 9 AiAOX, and 4 AlAOX gene family members. Phylogenetic, motif, domain, gene structure, and selective pressure analyses revealed that most AsAOXs, AiAOXs, and AlAOXs are evolutionarily conserved. We also identified 16 AsAOX segmental duplication pairs, suggesting that segmental duplication may have contributed to the expansion of the AsAOX gene family, potentially preserving these genes through subfunctionalization. Chromosome polyploidization, gene structural variations, and gene fragment recombination likely contributed to the evolution and expansion of the AsAOX gene family as well. Additionally, we hypothesize that AsAOX2 may have potential function in resisting wounding and heat stresses, while AsAOX4 could be specifically involved in mitigating wounding stress. AsAOX11 might contribute to resistance against chromium and waterlogging stresses. AsAOX8 may have potential fuction in mitigating ABA-mediated stress. AsAOX12 and AsAOX5 are most likely to have potential function in mitigating salt and drought stresses, respectively. This study elucidates the potential evolutionary pathways of the AsAOXs gene family, explores their responses and potential functions to various abiotic stresses, identifies potential candidate genes for future functional studies, and facilitates molecular breeding applications in A. sativa. Full article

The conversion of nitrate to ammonium, i.e., nitrate reduction, is a major consumer of reductants in plants. Previous studies have reported that the mitochondrial alternative oxidase (AOX) is upregulated under limited nitrate reduction conditions, including no/low nitrate or when ammonium is the sole nitrogen (N) source. Electron transfer from ubiquinone to AOX bypasses the proton-pumping complexes III and IV, thereby consuming reductants efficiently. Thus, upregulated AOX under limited nitrate reduction may dissipate excessive reductants and thereby attenuate oxidative stress. Nevertheless, so far there is no firm evidence for this hypothesis due to the lack of experimental systems to analyze the direct relationship between nitrate reduction and AOX. We therefore developed a novel culturing system for A. thaliana that manipulates shoot activities of nitrate reduction and AOX separately without causing N starvation, ammonium toxicity, or lack of nitrate signal. Using shoots processed with this system, we examined genome-wide gene expression and growth to better understand the relationship between AOX and nitrate reduction. The results showed that, only when nitrate reduction was limited, AOX deficiency significantly upregulated genes involved in mitochondrial oxidative stress, reductant shuttles, and non-phosphorylating bypasses of the respiratory chain, and inhibited growth. Thus, we conclude that AOX alleviates mitochondrial oxidative stress and sustains plant growth under limited nitrate reduction. Full article

Using Chlamydomonas as a model organism, we attempted to eliminate mitochondrial DNA (mtDNA) similar to rho⁰ or rho⁻ cells (completely or partially mtDNA-eliminated cells) in yeast. We successfully generated partially mtDNA-eliminated cells named as crm- cells, causing the inactivation of mitochondrial activity. We used three different chemicals to eliminate mtDNA including acriflavine (AF), ethidium bromide (EB) and dideoxycytidine (ddC) which prevents replication, inhibits POLG (DNA polymerase gamma) and terminates the mtDNA chain, respectively. The qPCR method was used to detect the mtDNA copy number and the selected rrnL6 gene for the detection of mitochondria, as well as the selected Chlamydomonas CC-124 strain. A reduction in the mitochondrial copy number led to a higher expression of AOX1, UCP1, PGRL1 and ICL1, which indicates the disturbance of the mitochondria–chloroplast ATP and NADPH balance. We selected AOX genes to further study this family and carried out a genome-wide search to identify AOX genes in green algae (C. reinhardtii). Our results revealed that C. reinhardtii contains four AOX genes, i.e., CrAOX1, CrAOX2, CrAOX3 and CrAOX4, which are distributed on Chr 3, Chr7 and Chr9. All CrAOX genes were predicted to localize in mitochondria using bioinformatics tools. Phylogenetic analysis suggests that these CrAOXs are subdivided into four groups and genes existing in the same group could perform identical functions. Collinearity analysis describes the strong evolutionary relationships of AOXs between the unicellular green algae Chlamydomonas reinhardtii and the multicellular green algae Volvox carteri. GO (gene ontology) annotation analysis predicted that CrAOXs played an integral part in carrying out alternate oxidative and respirative activities. Three putative miRNAs, cre-miR1162-3p, cre-miR1171 and cre-miR914, targeting the CrAOX2 gene were identified. Our studies have laid a foundation for the further use of partially mtDNA-eliminated cells and elucidating the functional characteristics of the AOX gene family. Full article

Catalases play a crucial role in the metabolism of reactive oxygen species (ROS) by converting H₂O₂ into molecular oxygen and water. They also contribute to virulence and fungal responses to various stresses. Previously, the MrCat1-deletion mutant (ΔMrCat1) was generated using the split-marker method in Metarhizium rileyi. In this study, the Cat1 gene was identified, and its function was evaluated. Under normal culture conditions, there were no significant differences in colony growth or dimorphic switching between ΔMrCat1 and the wild-type (WT) strains. However, under oxidative stress, the colony growth was inhibited, and the yeast–hyphal transition was suppressed in the ΔMrCat1 strain. Hyperosmotic stress did not differ significantly between the two strains. In the ΔMrCat1 strain, microsclerotia (MS) formation was delayed, resulting in less uniform MS size and a 76% decrease in MS yield compared to the WT strain. Moreover, the ΔMrCat1 strain exhibited diminished virulence. Gene expression analysis revealed up-regulation of ΔMrCat1, MrCat2, MrCat4, and MrAox in the ΔMrCat1 strain. These findings indicate that the MrCat1 gene in M. rileyi is essential for oxidative stress tolerance, MS formation, and virulence. Full article

The transcription of Arabidopsis organellar genes is performed by three nuclear-encoded RNA polymerases: RPOTm, RPOTmp, and RPOTp. The RPOTmp protein possesses ambiguous transit peptides, allowing participation in gene expression control in both mitochondria and chloroplasts, although its function in plastids is still under discussion. Here, we show that the overexpression of RPOTmp in Arabidopsis, targeted either to mitochondria or chloroplasts, disturbs the dormant seed state, and it causes the following effects: earlier germination, decreased ABA sensitivity, faster seedling growth, and earlier flowering. The germination of RPOTmp overexpressors is less sensitive to NaCl, while rpotmp knockout is highly vulnerable to salt stress. We found that mitochondrial dysfunction in the rpotmp mutant induces an unknown retrograde response pathway that bypasses AOX and ANAC017. Here, we show that RPOTmp transcribes the accD, clpP, and rpoB genes in plastids and up to 22 genes in mitochondria. Full article

In the post-harvest phase, fruit is inexorably subjected to extrinsic stressors that expedite energy expenditure and truncate the storage lifespan. The present study endeavors to elucidate the response strategies of litchi to the alterations of energy state caused by 2,4-Dinitrophenol (DNP) treatment through energy metabolism and sugar metabolism. It was observed that the DNP treatment reduced the energy state of the fruit, exacerbated membrane damage and triggered rapid browning in the pericarp after 24 h of storage. Furthermore, the expression of genes germane to energy metabolism (LcAtpB, LcAOX1, LcUCP1, LcAAC1, and, LcSnRK2) reached their peak within the initial 24 h of storage, accompanied by an elevation in the respiratory rate, which effectively suppressed the rise in browning index of litchi pericarp. The study also posits that, to cope with the decrease of energy levels and membrane damage, litchi may augment the concentrations of fructose, glucose, inositol, galactose, and sorbose, thus safeguarding the canonical metabolic functions of the fruit. Collectively, these findings suggest that litchi can modulate energy and sugar metabolism to cope with fruit senescence under conditions of energy deficiency. This study significantly advances the understanding of the physiological responses exhibited by litchi fruit to post-harvest external stressors. Full article

Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I’s assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing. Full article

Xylella fastidiosa (Xf) is a global bacterial threat for a diversity of plants, including olive trees. However, current understanding of host responses upon Xf-infection is limited to allow early disease prediction, diagnosis, and sustainable strategies for breeding on plant tolerance. Recently, we identified a major complex trait for early de novo programming, named CoV-MAC-TED, by comparing early transcriptome data during plant cell survival with SARS-CoV-2-infected human cells. This trait linked ROS/RNS balancing during first hours of stress perception with increased aerobic fermentation connected to alpha-tubulin-based cell restructuration and control of cell cycle progression. Furthermore, our group had advanced concepts and strategies for breeding on plant holobionts. Here, we studied tolerance against Xf-infection by applying a CoV-MAC-TED-related gene set to (1) progress proof-of-principles, (2) highlight the importance of individual host responses for knowledge gain, (3) benefit sustainable production of Xf-threatened olive, (4) stimulate new thinking on principle roles of secondary metabolite synthesis and microbiota for system equilibration and, (5) advance functional marker development for resilience prediction including tolerance to Xf-infections. We performed hypothesis-driven complex analyses in an open access transcriptome of primary target xylem tissues of naturally Xf-infected olive trees of the Xf-tolerant cv. Leccino and the Xf-susceptible cv. Ogliarola. The results indicated that cyanide-mediated equilibration of oxygen-dependent respiration and carbon-stress alleviation by the help of increased glycolysis-driven aerobic fermentation paths and phenolic metabolism associate to tolerance against Xf. Furthermore, enhanced alternative oxidase (AOX) transcript levels through transcription Gleichschaltung linked to quinic acid synthesis appeared as promising trait for functional marker development. Moreover, the results support the idea that fungal endophytes strengthen Xf-susceptible genotypes, which lack efficient AOX functionality. Overall, this proof-of-principles approach supports the idea that efficient regulation of the multi-functional AOX gene family can assist selection on multiple-resilience, which integrates Xf-tolerance, and stimulates future validation across diverse systems. Full article

During the period of ‘Feizixiao’ litchi fruit pericarp’s full coloring, there is a phenomenon of “sugar withdrawal” in the pulp, and the mixed foliar nutrients of calcium and magnesium (Ca+Mg) can effectively overcome this phenomenon. One of the reasons for this may be that it is related to the influence of the mixed nutrients of Ca+Mg on the bypass respiratory pathways of the pulp. The major fruit quality indicators, the rates of cytochrome and cyanide-resistant respiratory pathways (CP and AP) in the pulp and the activities of their key enzymes, were observed continuously in 2021 and 2022, and the deferentially expressed genes (DEGs) related to the two bypass respiratory pathways in the pulp were screened by RNA-seq analysis, with a qPCR of the random genes performed to verify the results. Ca+Mg treatment kept the content of the total soluble sugar in the pulp stable and higher than that the control in the ripening stage; Ca+Mg treatment increased the activities of electron-transferring enzymes in the electron transport chain, such as NADH dehydrogenase (ND), succinate dehydrogenase (SDH), cytochrome bc1 complex, and cytochrome c (Cyt c) through up-regulating their gene expression. In terms of the rate-limiting enzymes in the pulp, Ca+Mg treatment increased the activity of cytochrome oxidase (COX) in the CP pathway by up-regulating the expression of COX genes, then increased the CP respiratory rate and inhibited the CP respiratory rate decrease; meanwhile, it also inhibited the activity of AOX (alternate oxidase) in the pulp in the AP pathway by down-regulating the expression of AOX genes, then inhibited the increase in the AP respiration rate. The qPCR validation of randomly selected DEGs showed a significant unitary linear correlation between their expression levels and the results of the RNA-seq analysis. Therefore, one of the physiological mechanisms on the mixed foliar nutrients of Ca and Mg overcoming the phenomenon of “sugar withdrawal” in the ‘Feizixiao’ litchi pulp could be to promote CP and to inhibit AP, and then to delay the ripening and senescence of the pulp. Full article

Circadian rhythm disorders pose major risks to human health and animal production activity, and the hypothalamus is the center of circadian rhythm regulation. However, the epigenetic regulation of circadian rhythm based on farm animal models has been poorly investigated. We collected chicken hypothalamus samples at seven time points in one light/dark cycle and performed long noncoding RNA (lncRNA), circular RNA (circRNA), and mRNA sequencing to detect biomarkers associated with circadian rhythm. We enhanced the comprehensive expression profiling of ncRNAs and mRNAs in the hypothalamus and found two gene sets (circadian rhythm and retinal metabolism) associated with the light/dark cycle. Noncoding RNA networks with circadian expression patterns were identified by differential expression and circadian analysis was provided that included 38 lncRNAs, 15 circRNAs, and 200 candidate genes. Three lncRNAs (ENSGALT00000098661, ENSGALT00000100816, and MSTRG.16980.1) and one circRNA (novel_circ_010168) in the ncRNA–mRNA regulatory network were identified as key molecules influencing circadian rhythm by regulating AOX1 in retinal metabolism. These ncRNAs were predicted to be related to pernicious anemia, gonadal, eye disease and other disorders in humans. Together, the findings of this study provide insights into the epigenetic mechanisms of circadian rhythm and reveal AOX1 as a promising target of circadian rhythm regulation. Full article

In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOXα and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOXα-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future. Full article

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) (B. dorsalis) is an important agricultural, major invasive, and quarantine pest that can cause significant damage to the economic value of the fruit and vegetable industry. Male bait is one of the most effective methods of surveying, monitoring, and controlling B. dorsalis. In our study, we constructed cDNA libraries using total RNA extracted independently from the antennae, mouthparts, and thoracic legs of male and female adults and the ovipositors of female adults and screened out four aldehyde-oxidase-related genes (AOX-related), C58800, C66700, C67485, and C67698. Molecular docking predictions showed that eight compounds, including 3,4-dimethoxycinnamyl alcohol, 3,4-dimethoxy-cinnamaldehyde, deet, ethyl N-acetyl-N-butyl-β-alaninate, n-butyl butyrate, n-butyl butyrate, ethyl butyrate, methyl eugenol, and ethyl acetate, could combine with proteins encoded by the four B. dorsalis AOX-related genes. Furthermore, QPCR was performed to confirm that four compounds, including 3,4-dimethoxy cinnamic aldehyde, butyl levulinic acid ethyl ester (mosquito repellent), butyl butyrate, and methyl eugenol, induced significant changes in the AOX-related genes of B. dorsalis. These results provide useful information and guidance for the batch screening of potentially useful compounds and the search for effective attractants of B. dorsalis. Full article

Alternative oxidase (Aox) is a terminal oxidase operating in branched electron transport. The activity correlates positively with overflow metabolisms in certain Aspergilli, converting intracellular glucose by the shortest possible path into organic acids, like citrate or itaconate. Aox is nearly ubiquitous in fungi, but aox gene multiplicity is rare. Nevertheless, within the family of the Aspergillaceae and among its various species of industrial relevance—Aspergillus niger, A. oryzae, A. terreus, Penicillium rubens—paralogous aox genes coexist. Paralogous genes generally arise from duplication and are inherited vertically. Here, we provide evidence of four independent duplication events along the lineage that resulted in aox paralogues (aoxB) in contemporary Aspergillus and Penicillium taxa. In some species, three aox genes are co-expressed. The origin of the A. niger paralogue is different than that of the A. terreus paralogue, but all paralogous clades ultimately arise from ubiquitous aoxA parent genes. We found different patterns of uncorrelated gene losses reflected in the Aspergillus pedigree, albeit the original aoxA orthologues persist everywhere and are never replaced. The loss of acquired paralogues co-determines the contemporary aox gene content of individual species. In Aspergillus calidoustus, the two more ancient paralogues have, in effect, been replaced by two aoxB genes of distinct origins. Full article