Bc1 Complex Research Articles

Modifications of the respiratory chain of Bacillus licheniformis as an alkalophilic and cyanide-degrading microorganism.

Bacillus licheniformis can use cyanide as a nitrogen source for its growth. However, it can also carry out aerobic respiration in the presence of this compound, a classic inhibitor of mammalian cytochrome c oxidase, indicating that B. licheniformis has a branched respiratory chain with various terminal oxidases. Here, we studied the modifications in the respiratory chain of B. licheniformis when cells were cultured in Nutrient Broth, an alkaline medium with ammonium, or an alkaline medium with cyanide. Then, we measured oxygen consumption in intact cells and membranes, enzyme activities, carried out 1D and 2D-BN-PAGE, followed by mass spectrometry analysis of BN-PAGE bands associated with NADH, NADPH, and succinate dehydrogenase activities. We found that cell growth was favored in a nutrient medium than in an alkaline medium with cyanide. In parallel, respiratory activity progressively decreased in cells cultured in the rich medium, alkaline medium with ammonium, and the lowest activity was in the cells growing in the alkaline medium with cyanide. B. licheniformis membranes contain NADH, NADPH, and succinate dehydrogenases, and the proteomic analysis detected the nitrate reductase and the bc, caa3, aa3, and bd complexes. The succinate dehydrogenase migrated with a molecular mass of 375kDa, indicating its association with the nitrate reductase (115kDa + 241kDa, respectively). The NADH dehydrogenase of B. licheniformis forms aggregates of different molecular mass.

Synthesis, spectroscopic, computational, topology, and molecular docking studies on N,N'-(4-methyl-1,3-phenylene)bis(1-(2,4-dichlorophenyl)methanimine)

The study extensively examined N,N'-(4-methyl-1,3-phenylene)bis(1-(2,4-dichlorophenyl)methanimine) (6D). Advanced spectroscopic techniques, including IR, Raman, NMR, and UV-VIS spectroscopies, were employed to analyse the molecule. The Schiff base was ultimately confirmed using NMR spectrum analysis. The UV-VIS study revealed a notable bathochromic change in the compound, indicating their electronic transitions. By using the HOMO–LUMO bond gap the titled compound reactivity sites were identified. The compound 6D HOMO–LUMO band gap is 3.70 eV. Various wave function investigations like MESP, HOMO–LUMO, RDG, ELF, LOL, and ALIE were conducted to elucidate the distribution of electronic charge, providing valuable insights into the behaviour of molecules. The biological probability of the synthesised compound was extensively assessed using a docking study. Using MEP and HOMO–LUMO investigations, we confirm nucleophilic and electrophilic attacking sites. In the docking study, the protein Bovine cytochrome bc1 complex stigma Tellin bound (PDB ID–1PP9) was downloaded. The docking study identified the most stable minimum binding energy is –6.26 kcal/mol.

Architectures of photosynthetic RC-LH1 supercomplexes from Rhodobacter blasticus.

The reaction center-light-harvesting complex 1 (RC-LH1) plays an essential role in the primary reactions of bacterial photosynthesis. Here, we present high-resolution structures of native monomeric and dimeric RC-LH1 supercomplexes from Rhodobacter (Rba.) blasticus using cryo-electron microscopy. The RC-LH1 monomer is composed of an RC encircled by an open LH1 ring comprising 15 αβ heterodimers and a PufX transmembrane polypeptide. In the RC-LH1 dimer, two crossing PufX polypeptides mediate dimerization. Unlike Rhodabacter sphaeroides counterpart, Rba. blasticus RC-LH1 dimer has a less bent conformation, lacks the PufY subunit near the LH1 opening, and includes two extra LH1 αβ subunits, forming a more enclosed S-shaped LH1 ring. Spectroscopic assays reveal that these unique structural features are accompanied by changes in the kinetics of quinone/quinol trafficking between RC-LH1 and cytochrome bc1. Our findings reveal the assembly principles and structural variability of photosynthetic RC-LH1 supercomplexes, highlighting diverse strategies used by phototrophic bacteria to optimize light-harvesting and electron transfer in competitive environments.

Pyrimidine Azepine Targets the Plasmodium bc 1 Complex and Displays Multistage Antimalarial Activity.

Malaria control and elimination efforts would benefit from the identification and validation of new malaria chemotherapeutics. Recently, a transgenic Plasmodium berghei line was used to perform a series of high-throughput in vitro screens for new antimalarials acting against the parasite sexual stages. The screens identified pyrimidine azepine chemotypes with potent activity. Here, we validate the activity of PyAz90, the most potent pyrimidine azepine chemotype identified, against P. falciparum and P. vivax in the asexual and sexual stages. PyAz90 blocked parasite transmission to the mosquito vector at nanomolar concentrations and inhibited in vitro asexual parasite multiplication with a fast-action profile. Through the generation of P. falciparum PyAz90-resistant parasites and in vitro assays of mitochondrial activity, we identified cytochrome b as a molecular target of PyAz90. This work characterizes a promising chemotype that can be explored for the future development of new antimalarials targeting the Plasmodium cytochrome bc 1 complex.

Discovery of Novel Cyclobutyl Oxime Ester Derivatives Containing an α,β-Unsaturated Carbonyl Moiety as Potential Mitochondrial Toxins.

As part of continuous work to explore novel and efficient fungicides originating from natural products, a series of cyclobutyl oxime ester derivatives containing an α,β-unsaturated carbonyl moiety were designed and synthesized. In line with the primary evaluation of the inhibitory effect on common pathogenic fungi causing crop failure, a systematic study on the antifungal activity of target compounds against Rhizoctonia solani was carried out. Most target compounds exhibited satisfactory antifungal activity, and 10 of them were superior to the positive control trifloxystrobin. The most notable median effective concentration (EC50) of compound 6b was 1.70 μg/mL, which was considerable for an intensive study. The control efficacy of compound 6b on potted rice against R. solani was superior to trifloxystrobin at identical concentration. The mycelial morphology and cell membrane permeability of the treated fungi were disrupted, and the meaningful enzyme activities of SDH and POD were also restrained. The reactive oxygen species, nuclear morphology, and mitochondrial membrane potential of the treated hypha reflected an apparent difference compared with the normal morphology, which represented mitochondrial function damage. In addition, chemical features essential for the activity and docking mode within the compound and cytochrome bc1 complex were accessed by computer-aided technology. This study provided insights into the development of new green and efficient fungicides targeting the mitochondria.

Characterization of Shy1, the Schizosaccharomyces pombe homolog of human SURF1

Cytochrome c oxidase (complex IV) is the terminal enzyme in the mitochondrial respiratory chain. As a rare neurometabolic disorder caused by mutations in the human complex IV assembly factor SURF1, Leigh Syndrome (LS) is associated with complex IV deficiency. In this study, we comprehensively characterized Schizosaccharomyces pombe Shy1, the homolog of human SURF1. Bioinformatics analysis revealed that Shy1 contains a conserved SURF1 domain that links to the biogenesis of complex IV and shares high structural similarity with its homologs in Saccharomyces cerevisiae and humans. Our study showed that Shy1 is required for the expression of mtDNA-encoded genes and physically interacts with structural subunits and assembly factors of complex IV. Interestingly, Rip1, the subunit of ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (complex III), can also co-immunoprecipitate with Shy1, suggesting Shy1 may be involved in the assembly of the mitochondrial respiratory chain supercomplexes. This conclusion is further corroborated by our BN-PAGE analysis. Unlike its homologs, deletion of shy1 does not critically disrupt respiratory chain assembly, indicating the presence of the compensatory mechanism(s) within S. pombe that ensure mitochondrial functionality. Collectively, our investigation elucidates that Shy1 plays a pivotal role in the sustainability of the regular function of mitochondria by participating in the assembly of complex IV in S. pombe.

Discovery of 4-Hydroxyphenylpyruvate dioxygenase inhibitors with novel pharmacophores

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is pivotal in tyrosine metabolism and essential for plant survival. Its inhibition leads to leaf bleaching and plant death. While current HPPD inhibitors are effective, they pose phytotoxicity risks and may contribute to herbicide resistance. Here, we investigated the inhibitory potential of sethoxydim and atovaquone, which traditionally target acetyl-CoA carboxylase and the cytochrome bc1 complex, respectively. Both atovaquone and the degradation product of sethoxydim exhibited moderate HPPD inhibitory activity. But the mechanism by which sethoxydim inhibited HPPD remained unclear. Therefore, we embarked on an investigation into the crystal structure of the complex, with the aim of elucidating its precise binding mode. Our findings revealed that sethoxydim degrades in solution, producing dealkoxy sethoxydim as the active component in HPPD inhibition. Structural analysis elucidated the binding modes of atovaquone and dealkoxy sethoxydim with HPPD. These binding motifs represent novel pharmacophores and offer promising leads for developing HPPD inhibitors with improved pesticidal profiles.

Mechanisms of Short-Circuit Suppression in the bc1 Complex

Mechanisms of Short-Circuit Suppression in the bc1 Complex

Plasmodium falciparum Mitochondrial Complex III, the Target of Atovaquone, Is Essential for Progression to the Transmissible Sexual Stages.

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

Proton pathways and quantum effects in the Q-cycle of cytochrome bc1

Proton pathways and quantum effects in the Q-cycle of cytochrome bc1

Unveiling the potential: exploring the efficacy of complex III inhibitors in fungal disease control.

Phytopathogenic fungi are a key challenge to maximizing crop yield and quality for a growing global population. In this review, we give an overview of representative compounds that inhibit complex III, also known as bc1 complex, covering quinone inside inhibitors, quinone outside inhibitors, and quinone inside and outside inhibitors via the stigmatellin binding mode. Novel solutions to the escalating problem of resistance are still required, therefore compounds with alternative scaffolds, alternative docking modes, different mechanisms of action and improved efficacy against complex III necessitate ongoing research. © 2024 Society of Chemical Industry.

CRISPR/dCas12a knock-down of Acidithiobacillus ferrooxidans electron transport chain bc1 complexes enables enhanced metal sulfide bioleaching

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that plays an important role in biogeochemical iron and sulfur cycling and is a member of the consortia used in industrial hydrometallurgical processing of copper. Metal sulfide bioleaching is catalyzed by the regeneration of ferric iron, however, bioleaching of chalcopyrite, the dominant unmined form of copper on Earth, is inhibited by surface passivation. Here, we report the implementation of CRISPR interference (CRISPRi) using the catalytically inactive Cas12a (dCas12a) in A. ferrooxidans to knockdown the expression of genes in the petI and petII operons. These operons encode bc1 complex proteins and knockdown of these genes enabled the manipulation (enhancement or repression) of iron oxidation. The petB2 gene knockdown strain enhanced iron oxidation, leading to enhanced pyrite and chalcopyrite oxidation, which correlated with reduced biofilm formation and decreased surface passivation of the minerals. These findings highlight the utility of CRISPRi/dCas12a technology for engineering A. ferrooxidans while unveiling a new strategy to manipulate and improve bioleaching efficiency.

The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Synthesis and Structural Activity Relationship Study of Ursolic Acid Derivatives as Antitubercular Agent.

The chemical transformation of ursolic acid (UA) into novel C-3 aryl ester derivatives and in vitro and silico assessment of their antitubercular potential. UA is a natural pentacyclic triterpenoid with many pharmacological properties. Semisynthetic UA analogs have demonstrated enhanced anticancer, antimalarial, and antifilarial properties in our previous studies. The C-30 carboxylic group of previously isolated UA was protected, and various C-3 aryl ester derivatives were semi-synthesized. The agar dilution method was used to evaluate the in vitro antitubercular efficacy of Mycobacterium tuberculosis (Mtb) H37Ra. In silico docking studies of the active derivative were carried out against Mtb targets, catalase peroxidase (PDB: 1SJ2), dihydrofolate reductase (PDB: 4M2X), enoyl-ACP reductase (PDB: 4TRO), and cytochrome bc1 oxidase (PDB: 7E1V). The derivative 3-O-(2-amino,3-methyl benzoic acid)-ethyl ursolate (UA-1H) was the most active among the eight derivatives (MIC1 2.5 μg/mL) against Mtb H37Ra. Also, UA-1H demonstrated significant binding affinity in the range of 10.8-11.4 kcal/mol against the antiTb target proteins, which was far better than the positive control Isoniazid, Ethambutol, and co-crystallized ligand (HEM). Moreover, the predicted hit UA-1H showed no inhibition of Cytochrome P450 2D6 (CYP2D6), suggesting its potential for favorable metabolism in Phase I clinical studies. The ursolic acid derivative UA-1H possesses significant in vitro antitubercular potential with favorable in silico pharmacokinetics. Hence, further in vivo assessments are suggested for UA-1H for its possible development into a secure and efficient antitubercular drug.

Probing ligands to reaction centers to limit the photocycle in photosynthetic bacterium Rubrivivax gelatinosus

Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view. SignificanceA crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)−1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.

New synergistic benzoquinone scaffolds as inhibitors of mycobacterial cytochrome bc1 complex to treat multi-drug resistant tuberculosis

Through a comprehensive molecular docking study, a unique series of naphthoquinones clubbed azetidinone scaffolds was arrived with promising binding affinity to Mycobacterial Cytbc1 complex, a drug target chosen to kill multi-drug resistant Mycobacterium tuberculosis (MDR-Mtb). Five compounds from series-2, 2a, 2c, 2g, 2h, and 2j, showcased significant in vitro anti-tubercular activities against Mtb H37Rv and MDR clinical isolates. Further, synergistic studies of these compounds in combination with INH and RIF revealed a potent bactericidal effect of compound 2a at concentration of 0.39 μg/mL, and remaining (2c, 2g, 2h, and 2j) at 0.78 μg/mL. Exploration into the mechanism study through chemo-stress assay and proteome profiling uncovered the down-regulation of key proteins of electron-transport chain and Cytbc1 inhibition pathway. Metabolomics corroborated these proteome findings, and heightened further understanding of the underlying mechanism. Notably, in vitro and in vivo animal toxicity studies demonstrated minimal toxicity, thus underscoring the potential of these compounds as promising anti-TB agents in combination with RIF and INH. These active compounds adhered to Lipinski's Rule of Five, indicating the suitability of these compounds for drug development. Particular significance of molecules NQ02, 2a, and 2h, which have been patented (Published 202141033473).

Bactericidal and sterilizing activity of sudapyridine-clofazimine-TB47 combined with linezolid or pyrazinamide in a murine model of tuberculosis.

As an obligate aerobe, Mycobacterium tuberculosis relies on its branched electron transport chain (ETC) for energy production through oxidative phosphorylation. Regimens targeting ETC exhibit promising potential to enhance bactericidal activity against M. tuberculosis and hold the prospect of shortening treatment duration. Our previous research demonstrated that the bacteriostatic drug candidate TB47 (T) inhibited the growth of M. tuberculosis by targeting the cytochrome bc1 complex and exhibited synergistic activity with clofazimine (C). Here, we found synergistic activities between C and sudapyridine (S), a structural analog of bedaquiline (B). S has shown similar anti-tuberculosis efficacy and may share a mechanism of action with B, which inhibits ATP synthesis and the energy metabolism of bacteria. We evaluated the efficacy of SCT in combination with linezolid (L) or pyrazinamide (Z) using a well-established murine model of tuberculosis. Compared to the BPa(pretomanid)L regimen, SCT and SCTL demonstrated similar bactericidal and sterilizing activities. There was no significant difference in activity between SCT and SCTL. In contrast, SCZ and SCTZ showed much higher activities, with none of the 15 mice experiencing relapse after 2 months of treatment with either SCZ or SCTZ. However, T did not contribute to the activity of the SCZ. Our findings emphasize the efficacy and the potential clinical significance of combination therapy with ETC inhibitors. Additionally, cross-resistance exists not only between S and B but also between S/B and C. This is supported by our findings, as spontaneous S-resistant mutants exhibited mutations in Rv0678, which are associated with cross-resistance to B and C.

Effectiveness of Two New Endochin-like Quinolones, ELQ-596 and ELQ-650, in Experimental Mouse Models of Human Babesiosis.

Endochin-like quinolones (ELQs) define a class of small molecule antimicrobials that target the mitochondrial electron transport chain of various human parasites by inhibiting their cytochrome bc1 complexes. The compounds have shown potent activity against a wide range of protozoan parasites, including the intraerythrocytic parasites Plasmodium and Babesia, the agents of human malaria and babesiosis, respectively. First-generation ELQ compounds were previously found to reduce infection by Babesia microti and Babesia duncani in animal models of human babesiosis but achieved a radical cure only in combination with atovaquone and required further optimization to address pharmacological limitations. Here, we report the identification of two second-generation 3-biaryl ELQ compounds, ELQ-596 and ELQ-650, with potent antibabesial activity in vitro and favorable pharmacological properties. In particular, ELQ-598, a prodrug of ELQ-596, demonstrated high efficacy as an orally administered monotherapy at 10 mg/kg. The compound achieved radical cure in both the chronic model of B. microti-induced babesiosis in immunocompromised mice and the lethal infection model induced by B. duncani in immunocompetent mice. Given its high potency, favorable physicochemical properties, and low toxicity profile, ELQ-596 represents a promising drug for the treatment of human babesiosis.

Exploring Scaffold Hopping for Novel 2-(Quinolin-4-yloxy)acetamides with Enhanced Antimycobacterial Activity.

Utilizing a scaffold-hopping strategy from the drug candidate telacebec, a novel series of 2-(quinolin-4-yloxy)acetamides was synthesized and evaluated as inhibitors of Mycobacterium tuberculosis (Mtb) growth. These compounds demonstrated potent activity against drug-sensitive and multidrug-resistant strains (MIC ≤ 0.02 μM). Leading compounds were evaluated against a known qcrB resistant strain (T313A), and their loss in activity suggested that the cytochrome bc1 complex is the likely target. Additionally, these structures showed high selectivity regarding mammalian cells (selectivity index > 500) and stability across different aqueous media. Furthermore, some of the synthesized quinolines demonstrated aqueous solubility values that exceeded those of telacebec, while maintaining low rates of metabolism. Finally, a selected compound prevented Mtb growth by more than 1.7 log10 colony forming units in a macrophage model of tuberculosis (TB) infection. These findings validate the proposed design and introduce new 2-(quinolin-4-yloxy)acetamides with potential for development in TB drug discovery campaigns.

The Effects of Mixed Foliar Nutrients of Calcium and Magnesium on the Major Bypass Respiratory Pathways in the Pulp of ‘Feizixiao’ Litchi

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.