Network pharmacology and molecular docking reveal the immunomodulatory mechanism of rhubarb peony decoction for the treatment of ulcerative colitis and irritable bowel syndrome.

Background: Ulcerative colitis (UC) and irritable bowel syndrome (IBS) share various similarities in clinical symptoms, pathogenesis, and treatment. UC concurrent IBS tends toward more severe symptoms and worse prognosis, and promising feasible therapies for the overlapping symptoms remains a challenge. Rhubarb peony decoction (RPD) is a well-known traditional Chinese medicine that has been widely applied in treating UC. RPD may exert extensive therapeutic effects on both IBS and UC. However, the common mechanism of its treatment remains unclear. We aimed to assess the potential pharmacological mechanism of RPD in the treatment of overlapping IBS and UC. Methods: The active components and targets of RPD were retrieved from ETCM, TCMSP, BATMAN-TCM, and TCM databases. The disease targets were screened by searching the DrugBank, OMIM, TTD, and PharmGKB databases. PPI network analysis was performed and visualized via the STRING platform and Cytoscape software. GO and KEGG enrichment analyses of the hub genes of RPD were predicted to elucidate the potential molecular mechanism. Subsequently, molecular docking was carried out to verify the combination of active compounds with core targets. Results: By integrating all targets of RPD and disease, a total of 31 bioactive ingredients were identified including quercetin, kaempferol, aloe-emodin, beta-sitosterol, and (+)-catechin, etc. JUN, TP53, MAPK1, RELA, MYC, and ESR1 were explored as potential therapeutic targets among 126 common drug-disease-related targets. They were enriched in the AGE-RAGE signaling pathway in diabetic complications, as well as the NF-kappa B signaling pathway and MAPK signaling pathway. Additionally, some active ingredients were identified as candidates for binding to the hub targets via molecular docking, further suggesting their anti-inflammatory and antioxidative properties. Conclusion: RPD may exert the overall treatment effect for UC and IBS overlap syndrome via the biological mechanism of "multi-ingredients, multi-targets, and multi-pathways" on inflammation, oxidative stress, immune, oncogenicity, and gut microbiota dysbiosis.

Ligand-induced activation and G protein coupling of prostaglandin F2α receptor.

Prostaglandin F2α (PGF2α), an endogenous arachidonic acid metabolite, regulates diverse physiological functions in many tissues and cell types through binding and activation of a G-protein-coupled receptor (GPCR), the PGF2α receptor (FP), which also is the primary therapeutic target for glaucoma and several other diseases. Here, we report cryo-electron microscopy (cryo-EM) structures of the human FP bound to endogenous ligand PGF2α and anti-glaucoma drugs LTPA and TFPA at global resolutions of 2.67 Å, 2.78 Å, and 3.14 Å. These structures reveal distinct features of FP within the lipid receptor family in terms of ligand binding selectivity, its receptor activation, and G protein coupling mechanisms, including activation in the absence of canonical PIF and ERY motifs and Gq coupling through direct interactions with receptor transmembrane helix 1 and intracellular loop 1. Together with mutagenesis and functional studies, our structures reveal mechanisms of ligand recognition, receptor activation, and G protein coupling by FP, which could facilitate rational design of FP-targeting drugs.

Improving diameter measurement from Fraunhofer diffraction with fringe segment splicing.

Fraunhofer diffraction is an easy but powerful method for measuring the diameter of a thin filament. In practice, however, the diffraction pattern attainable is always subject to limits imposed by various imperfections in real systems, such as small angle approximation and sensor threshold, thus degrading the measurement resolution. In this Letter, we propose a method of fringe segment splicing for improving the diameter measurement from Fraunhofer diffraction. The fringe segment is chosen from a real diffraction pattern and is used to reproduce an ideal diffraction fringe, where the theoretical estimates give the best approximation to the observations. The problem of diameter measurement is solved in the spatial frequency-domain with an ideal diffraction fringe. Our results show that the relative error in this method is less than 0.1% and is far superior to that of previous methods.

Estimating divergent forest carbon stocks and sinks via a knife set approach.

Forest carbon stocks and sinks (CSSs) have been widely estimated using climate classification tables and linear regression (LR) models with common independent variables (IVs) such as the average diameter at breast height (DBH) of stems and root shoot ratio. However, this approach is relatively ineffective when the explanatory power of IVs is lower than that of unobservable variables. Various environmental and anthropogenic factors affect target variables that cause the correlation between them to be chaotic. Here, we designed a knife set (KS) approach combining LR models and the wandering through random forests (WTF) algorithm and applied it in a specific case of Phyllostachys edulis (Carrière) J. Houz. (P. edulis) forests, which have an irregular relationship between their belowground carbon (BGC) stocks and average DBH. We then validated the KS approach performed by cluster computing to estimate the aboveground carbon (AGC) and BGC stocks and the total net primary production (TNPP). The estimated CSSs were compared to the benchmark of the methodology that applied Tier 1 in the Intergovernmental Panel on Climate Change (IPCC) Guidelines for National Greenhouse Gas Inventories via 10-fold cross validation, and the KS approach significantly increased precision and accuracy of estimations. Our approach provides general insights to accurately estimate forest CSSs relying on evidence-based field data, even if some target variables are divergent in specific forest types. We also pointed out the reason why current fancy models containing machine learning (ML) or deep learning algorithms are not effective in predicting the target variables of certain chaotic systems is perhaps that the total explanatory power of observable variables is less than that of the total unobservable variables. Quantifying unobservable variables into observable variables is a linchpin of future works related to chaotic system estimation.

Structure of PDE3A-SLFN12 complex and structure-based design for a potent apoptosis inducer of tumor cells.

Molecular glues are a class of small molecular drugs that mediate protein-protein interactions, that induce either the degradation or stabilization of target protein. A structurally diverse group of chemicals, including 17-ß-estradiol (E2), anagrelide, nauclefine, and DNMDP, induces apoptosis by forming complexes with phosphodiesterase 3A (PDE3A) and Schlafen 12 protein (SLFN12). They do so by binding to the PDE3A enzymatic pocket that allows the compound-bound PDE3A to recruit and stabilize SLFN12, which in turn blocks protein translation, leading to apoptosis. In this work, we report the high-resolution cryo-electron microscopy structure of PDE3A-SLFN12 complexes isolated from cultured HeLa cells pre-treated with either anagrelide, or nauclefine, or DNMDP. The PDE3A-SLFN12 complexes exhibit a butterfly-like shape, forming a heterotetramer with these small molecules, which are packed in a shallow pocket in the catalytic domain of PDE3A. The resulting small molecule-modified interface binds to the short helix (E552-I558) of SLFN12 through hydrophobic interactions, thus "gluing" the two proteins together. Based on the complex structure, we designed and synthesized analogs of anagrelide, a known drug used for the treatment of thrombocytosis, to enhance their interactions with SLFN12, and achieved superior efficacy in inducing apoptosis in cultured cells as well as in tumor xenografts.

A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin2α-induced corpus luteum regression.

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.

Casein kinase 1G2 suppresses necroptosis-promoted testis aging by inhibiting receptor-interacting kinase 3.

Casein kinases are a large family of intracellular serine/threonine kinases that control a variety of cellular signaling functions. Here we report that a member of casein kinase 1 family, casein kinase 1G2, CSNK1G2, binds and inhibits the activation of receptor-interacting kinase 3, RIPK3, thereby attenuating RIPK3-mediated necroptosis. The binding of CSNK1G2 to RIPK3 is triggered by auto-phosphorylation at serine 211/threonine 215 sites in its C-terminal domain. CSNK1G2-knockout mice showed significantly enhanced necroptosis response and premature aging of their testis, a phenotype that was rescued by either double knockout of the Ripk3 gene or feeding the animal with a RIPK1 kinase inhibitor-containing diet. Moreover, CSNK1G2 is also co-expressed with RIPK3 in human testis, and the necroptosis activation marker phospho-MLKL was observed in the testis of old (>80) but not young men, indicating that the testis-aging program carried out by the RIPK3-mediated and CSNK1G2-attenuated necroptosis is evolutionarily conserved between mice and men.

Genome-wide identification of the restorer-of-fertility-like (RFL) gene family in Brassica napus and expression analysis in Shaan2A cytoplasmic male sterility.

BACKGROUND: Cytoplasmic male sterility (CMS) is very important in hybrid breeding. The restorer-of-fertility (Rf) nuclear genes rescue the sterile phenotype. Most of the Rf genes encode pentatricopeptide repeat (PPR) proteins. RESULTS: We investigated the restorer-of-fertility-like (RFL) gene family in Brassica napus. A total of 53 BnRFL genes were identified. While most of the BnRFL genes were distributed on 10 of the 19 chromosomes, gene clusters were identified on chromosomes A9 and C8. The number of PPR motifs in the BnRFL proteins varied from 2 to 19, and the majority of BnRFL proteins harbored more than 10 PPR motifs. An interaction network analysis was performed to predict the interacting partners of RFL proteins. Tissue-specific expression and RNA-seq analyses between the restorer line KC01 and the sterile line Shaan2A indicated that BnRFL1, BnRFL5, BnRFL6, BnRFL8, BnRFL11, BnRFL13 and BnRFL42 located in gene clusters on chromosomes A9 and C8 were highly expressed in KC01. CONCLUSIONS: In the present study, identification and gene expression analysis of RFL gene family in the CMS system were conducted, and seven BnRFL genes were identified as candidates for the restorer genes in Shaan2A CMS. Taken together, this method might provide new insight into the study of Rf genes in other CMS systems.

An alkaloid initiates phosphodiesterase 3A-schlafen 12 dependent apoptosis without affecting the phosphodiesterase activity.

The promotion of apoptosis in tumor cells is a popular strategy for developing anti-cancer drugs. Here, we demonstrate that the plant indole alkaloid natural product nauclefine induces apoptosis of diverse cancer cells via a PDE3A-SLFN12 dependent death pathway. Nauclefine binds PDE3A but does not inhibit the PDE3A's phosphodiesterase activity, thus representing a previously unknown type of PDE3A modulator that can initiate apoptosis without affecting PDE3A's canonical function. We demonstrate that PDE3A's H840, Q975, Q1001, and F1004 residues-as well as I105 in SLFN12-are essential for nauclefine-induced PDE3A-SLFN12 interaction and cell death. Extending these molecular insights, we show in vivo that nauclefine inhibits tumor xenograft growth, doing so in a PDE3A- and SLFN12-dependent manner. Thus, beyond demonstrating potent cytotoxic effects of an alkaloid natural product, our study illustrates a potentially side-effect-reducing strategy for targeting PDE3A for anti-cancer therapeutics without affecting its phosphodiesterase activity.

Estrogen-Related Hormones Induce Apoptosis by Stabilizing Schlafen-12 Protein Turnover.

The mitochondrial pathway of apoptosis is controlled by the ratio of anti- and pro-apoptotic members of the Bcl-2 family of proteins. The molecular events underlying how a given physiological stimulus changes this ratio to trigger apoptosis remains unclear. We report here that human 17-ß-estradiol (E2) and its related steroid hormones induce apoptosis by binding directly to phosphodiesterase 3A, which in turn recruits and stabilizes an otherwise fast-turnover protein Schlafen 12 (SLFN12). The elevated SLFN12 binds to ribosomes to exclude the recruitment of signal recognition particles (SRPs), thereby blocking the continuous protein translation occurring on the endoplasmic reticulum of E2-treated cells. These proteins include Bcl-2 and Mcl-1, whose ensuing decrease triggers apoptosis. The SLFN12 protein and an apoptosis activation marker were co-localized in syncytiotrophoblast of human placentas, where levels of estrogen-related hormones are high, and dynamic cell turnover by apoptosis is critical for successful implantation and placenta development.

Transcriptomic and Proteomic Analysis of Shaan2A Cytoplasmic Male Sterility and Its Maintainer Line in Brassica napus.

Cytoplasmic male sterility (CMS) lines are widely used for hybrid production in Brassica napus. The Shaan2A CMS system is one of the most important in China and has been used for decades; however, the male sterility mechanism underlying Shaan2A CMS remains unknown. Here, we performed transcriptomic and proteomic analysis, combined with additional morphological observation, in the Shaan2A CMS. Sporogenous cells, endothecium, middle layer, and tapetum could not be clearly distinguished in Shaan2A anthers. Furthermore, Shaan2A anther chloroplasts contained fewer starch grains than those in Shaan2B (a near-isogenic line of Shaan2A), and the lamella structure of chloroplasts in Shaan2A anther wall cells was obviously aberrant. Transcriptomic analysis revealed differentially expressed genes (DEGs) mainly related to carbon metabolism, lipid and flavonoid metabolism, and the mitochondrial electron transport/ATP synthesis pathway. Proteomic results showed that differentially expressed proteins were mainly associated with carbohydrate metabolism, energy metabolism, and genetic information processing pathways. Importantly, nine gene ontology categories associated with anther and pollen development were enriched among down-regulated DEGs at the young bud (YB) stage, including microsporogenesis, sporopollenin biosynthetic process, and tapetal layer development. Additionally, 464 down-regulated transcription factor (TF) genes were identified at the YB stage, including some related to early anther differentiation such as SPOROCYTELESS (SPL, also named NOZZLE, NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), MYB80 (formerly named MYB103), and ABORTED MICROSPORES (AMS). These results suggested that the sterility gene in the Shaan2A mitochondrion might suppress expression of these TF genes in the nucleus, affecting early anther development. Finally, we constructed an interaction network of candidate proteins based on integrative analysis. The present study provides new insights into the molecular mechanism of Shaan2A CMS in B. napus.

Genetic dissection of the mechanism of flowering time based on an environmentally stable and specific QTL in Brassica napus.

Flowering time is an important agronomic trait that is highly influenced by the environment. To elucidate the genetic mechanism of flowering time in rapeseed (Brassica napus L.), a genome-wide QTL analysis was performed in a doubled haploid population grown in winter, semi-winter and spring ecological conditions. Fifty-five consensus QTLs were identified after combining phenotype and genomic data, including 12 environment-stable QTLs and 43 environment-specific QTLs. Importantly, six major QTLs for flowering time were identified, of which two were considered environment-specific QTLs in spring ecological condition and four were considered environment-stable QTLs in winter and semi-winter ecological conditions. Through QTL comparison, 18 QTLs were colocalized with QTLs from six other published studies. Combining the candidate genes with their functional annotation, in 49 of 55 consensus QTLs, 151 candidate genes in B. napus corresponding to 95 homologous genes in Arabidopsis thaliana related to flowering were identified, including BnaC03g32910D (CO), BnaA02g12130D (FT) and BnaA03g13630D (FLC). Most of the candidate genes were involved in different flowering regulatory pathways. Based on re-sequencing and differences in sequence annotation between the two parents, we found that regions containing some candidate genes have numerous non-frameshift InDels and many non- synonymous mutations, which might directly lead to gene functional variation. Flowering time was negativly correlated with seed yield and thousand seed weight based on a QTL comparison of flowering time and seed yield traits, which has implications in breeding new early-maturing varieties of B. napus. Moreover, a putative flowering regulatory network was constructed, including the photoperiod, circadian clock, vernalization, autonomous and gibberellin pathways. Multiple copies of genes led to functional difference among the different copies of homologous genes, which also increased the complexity of the flowering regulatory networks. Taken together, the present results not only provide new insights into the genetic regulatory network underlying the control of flowering time but also improve our understanding of flowering time regulatory pathways in rapeseed.

RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system.

A pair of kinases, RIPK1 and RIPK3, as well as the RIPK3 substrate MLKL cause a form of programmed necrotic cell death in mammals termed necroptosis. We report here that male reproductive organs of both Ripk3- and Mlkl-knockout mice retain 'youthful' morphology and function into advanced age, while those of age-matched wild-type mice deteriorate. The RIPK3 phosphorylation of MLKL, the activation marker of necroptosis, is detected in spermatogonial stem cells in the testes of old but not in young wild-type mice. When the testes of young wild-type mice are given a local necroptotic stimulus, their reproductive organs showed accelerated aging. Feeding of wild-type mice with an RIPK1 inhibitor prior to the normal onset of age-related changes in their reproductive organs blocked the appearance of signs of aging. Thus, necroptosis in testes promotes the aging-associated deterioration of the male reproductive system in mice.

Natural Product Kongensin A is a Non-Canonical HSP90 Inhibitor that Blocks RIP3-dependent Necroptosis.

RIP3-dependent necroptosis has recently garnered significant interest because of the unique signaling mechanisms and pathologic functions involved in this process. Accordingly, a number of chemical screens have identified several effective small-molecule inhibitors that specifically block necroptosis. Here, we report the discovery that kongensin A (KA), a natural product isolated from Croton kongensis, is a potent inhibitor of necroptosis and an inducer of apoptosis. Using a new bioorthogonal ligation method (TQ ligation), we reveal that the direct cellular target of KA is heat shock protein 90 (HSP90). Further studies demonstrate that KA covalently binds to a previously uncharacterized cysteine 420 in the middle domain of HSP90 and dissociates HSP90 from its cochaperone CDC37, which leads to inhibition of RIP3-dependent necroptosis and promotion of apoptosis in multiple cancer cell lines. Collectively, our findings demonstrate that KA is an effective HSP90 inhibitor that has potential anti-necroptosis and anti-inflammation applications.

Identification of the Relationship between Oil Body Morphology and Oil Content by Microstructure Comparison Combining with QTL Analysis in Brassica napus.

Oil bodies (OBs) are relatively simple but very important organelles comprising a matrix of triacylglycerol (TAG) surrounded by a phospholipid monolayer embedded and covered with unique proteins. The OB structure in Brassica napus with different oil content and the relationship between the oil content and the OB structure needs to be better understood. In this paper, the characteristics of OBs in the embryo of a series of B. napus materials with different oil content ranging from 34% to over 60% were studied. The results indicated that the OB size was significantly positively correlated with the oil content but was significantly negatively correlated with the glucosinolates and the protein content. Many genes associated with TAG synthesis, OB-membrane proteins, and the cell progress regulatory pathway were identified in the confidence interval of co-located QTLs for oil content, fatty acid (FA) compositions, and protein content. Our results suggested that the morphology of OBs might be directly controlled by the genes associated with OB-membrane proteins and indirectly controlled by the genes associated with TAG synthesis and cell progress regulatory pathway.

A cytosolic heat shock protein 90 and cochaperone CDC37 complex is required for RIP3 activation during necroptosis.

Receptor-interacting protein kinase 3, RIP3, and a pseudokinase mixed lineage kinase-domain like protein, MLKL, constitute the core components of the necroptosis pathway, which causes programmed necrotic death in mammalian cells. Latent RIP3 in the cytosol is activated by several upstream signals including the related kinase RIP1, which transduces signals from the tumor necrosis factor (TNF) family of cytokines. We report here that RIP3 activation following the induction of necroptosis requires the activity of an HSP90 and CDC37 cochaperone complex. This complex physically associates with RIP3. Chemical inhibitors of HSP90 efficiently block necroptosis by preventing RIP3 activation. Cells with knocked down CDC37 were unable to respond to necroptosis stimuli. Moreover, an HSP90 inhibitor that is currently under clinical development as a cancer therapy was able to prevent systemic inflammatory response syndrome in rats treated with TNF-α. HSP90 and CDC37 cochaperone complex-mediated protein folding is thus an important part of the RIP3 activation process during necroptosis.

Quantitative trait loci that control the oil content variation of rapeseed (Brassica napus L.).

KEY MESSAGE: This report describes an integrative analysis of seed-oil-content quantitative trait loci (QTL) in Brassica napus , using a high-density genetic map to align QTL among different populations. Rapeseed (Brassica napus) is an important source of edible oil and sustainable energy. Given the challenge involved in using only a few genes to substantially increase the oil content of rapeseed without affecting the fatty acid composition, exploitation of a greater number of genetic loci that regulate the oil content variation among rapeseed germplasm is of fundamental importance. In this study, we investigated variation in the seed-oil content among two related genetic populations of Brassica napus, the TN double-haploid population and its derivative reconstructed-F2 population. Each population was grown in multiple experiments under different environmental conditions. Mapping of quantitative trait loci (QTL) identified 41 QTL in the TN populations. Furthermore, of the 20 pairs of epistatic interaction loci detected, approximately one-third were located within the QTL intervals. The use of common markers on different genetic maps and the TN genetic map as a reference enabled us to project QTL from an additional three genetic populations onto the TN genetic map. In summary, we used the TN genetic map of the B. napus genome to identify 46 distinct QTL regions that control seed-oil content on 16 of the 19 linkage groups of B. napus. Of these, 18 were each detected in multiple populations. The present results are of value for ongoing efforts to breed rapeseed with high oil content, and alignment of the QTL makes an important contribution to the development of an integrative system for genetic studies of rapeseed.

Identification of QTLs associated with oil content in a high-oil Brassica napus cultivar and construction of a high-density consensus map for QTLs comparison in B. napus.

Increasing seed oil content is one of the most important goals in breeding of rapeseed (B. napus L.). To dissect the genetic basis of oil content in B. napus, a large and new double haploid (DH) population containing 348 lines was obtained from a cross between 'KenC-8' and 'N53-2', two varieties with >10% difference in seed oil content, and this population was named the KN DH population. A genetic linkage map consisting of 403 markers was constructed, which covered a total length of 1783.9 cM with an average marker interval of 4.4 cM. The KN DH population was phenotyped in eight natural environments and subjected to quantitative trait loci (QTL) analysis for oil content. A total of 63 identified QTLs explaining 2.64-17.88% of the phenotypic variation were identified, and these QTLs were further integrated into 24 consensus QTLs located on 11 chromosomes using meta-analysis. A high-density consensus map with 1335 marker loci was constructed by combining the KN DH map with seven other published maps based on the common markers. Of the 24 consensus QTLs in the KN DH population, 14 were new QTLs including five new QTLs in A genome and nine in C genome. The analysis revealed that a larger population with significant differences in oil content gave a higher power detecting new QTLs for oil content, and the construction of the consensus map provided a new clue for comparing the QTLs detected in different populations. These findings enriched our knowledge of QTLs for oil content and should be a potential in marker-assisted breeding of B. napus.

Proteomic and comparative genomic analysis of two Brassica napus lines differing in oil content.

Ultrastructural observations, combined with proteomic and comparative genomic analyses, were applied to interpret the differences in protein composition and oil-body characteristics of mature seed of two Brassica napus lines with high and low oil contents of 55.19% and 36.49%, respectively. The results showed that oil bodies were arranged much closer in the high than in the low oil content line, and differences in cell size and thickness of cell walls were also observed. There were 119 and 32 differentially expressed proteins (DEPs) of total and oil-body proteins identified. The 119 DEPs of total protein were mainly involved in the oil-related, dehydration-related, storage and defense/disease, and some of these may be related to oil formation. The DEPs involved with dehydration-related were both detected in total and oil-body proteins for high and low oil lines and may be correlated with the number and size of oil bodies in the different lines. Some genes that corresponded to DEPs were confirmed by quantitative trait loci (QTL) mapping analysis for oil content. The results revealed that some candidate genes deduced from DEPs were located in the confidence intervals of QTL for oil content. Finally, the function of one gene that coded storage protein was verified by using a collection of Arabidopsis lines that can conditionally express the full length cDNA from developing seeds of B. napus.

Involvement of alternative oxidase in the regulation of sensitivity of Sclerotinia sclerotiorum to the fungicides azoxystrobin and procymidone.

Sclerotinia sclerotiorum is a filamentous fungal pathogen that can infect many economically important crops and vegetables. Alternative oxidase is the terminal oxidase of the alternative respiratory pathway in fungal mitochondria. The function of alternative oxidase was investigated in the regulation of sensitivity of S. sclerotiorum to two commercial fungicides, azoxystrobin and procymidone which have different fungitoxic mechanisms. Two isolates of S. sclerotiorum were sensitive to both fungicides. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, significantly increased the values of effective concentration causing 50% mycelial growth inhibition (EC50) of azoxystrobin to both S. sclerotiorum isolates, whereas notably decreased the EC50 values of procymidone. In mycelial respiration assay azoxystrobin displayed immediate inhibitory effect on cytochrome pathway capacity, but had no immediate effect on alternative pathway capacity. In contrast, procymidone showed no immediate impact on capacities of both cytochrome and alternative pathways in the mycelia. However, alternative oxidase encoding gene (aox) transcript and protein levels, alternative respiration pathway capacity of the mycelia were obviously increased by pre-treatment for 24 h with both azoxystrobin and procymidone. These results indicate that alternative oxidase was involved in the regulation of sensitivity of S. sclerotiorum to the fungicides azoxystrobin and procymidone, and that both fungicides could affect aox gene expression and the alternative respiration pathway capacity development in mycelia of this fungal pathogen.