WUSCHEL-related homeobox transcription factor SlWOX13 regulates tomato fruit ripening.

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established importance of WUSCHEL-related homeobox (WOX) TFs in plant development, the involvement of WOX and its underlying mechanism in the regulation of fruit ripening remain unclear. Here, we demonstrate that SlWOX13 regulates fruit ripening in tomato (Solanum lycopersicum). Overexpression of SlWOX13 accelerates fruit ripening, whereas loss-of-function mutation in SlWOX13 delays this process. Moreover, ethylene synthesis and carotenoid accumulation are significantly inhibited in slwox13 mutant fruit but accelerated in SlWOX13 transgenic fruit. Integrated analyses of RNA-seq and chromatin immunoprecipitation (ChIP)-seq identified 422 direct targets of SlWOX13, of which 243 genes are negatively regulated and 179 are positively regulated by SlWOX13. Electrophoretic mobility shift assay, RT-qPCR, dual-luciferase reporter assay, and ChIP-qPCR analyses demonstrated that SlWOX13 directly activates the expression of several genes involved in ethylene synthesis and signaling and carotenoid biosynthesis. Furthermore, SlWOX13 modulates tomato fruit ripening through key ripening-related TFs, such as RIPENING INHIBITOR (RIN), NON-RIPENING (NOR), and NAM, ATAF1, 2, and CUC2 4 (NAC4). Consequently, these effects promote fruit ripening. Taken together, these results demonstrate that SlWOX13 positively regulates tomato fruit ripening via both ethylene synthesis and signaling and by transcriptional regulation of key ripening-related TFs.

Histone H3K27 Demethylase SlJMJ3 Modulates Fruit Ripening in Tomato.

The histone lysine (K) demethylase 4 (KDM4/JHDM3) subfamily of jumonji domain-containing demethylases (JMJs) has been implicated in various aspects of plant development. However, their involvement in regulating the ripening of fleshy fruits remains unclear. Here, we identified SlJMJ3, a member of the KDM4/JHDM3 family, as a H3K27me3 demethylase in tomato (Solanum lycopersicum) that plays an important role in fruit ripening regulation. Overexpression of SlJMJ3 led to accelerated fruit ripening, whereas loss-of-function of SlJMJ3 delayed this process. Furthermore, we determined that SlJMJ3 exerts its regulatory function by modulating the expression of multiple ripening-related genes involved in ethylene biosynthesis and response, carotenoid metabolism, cell wall modification, transcriptional control, and DNA methylation modification. SlJMJ3 bound directly to the promoters of ripening-related genes harboring the CTCTGYTY motif and activates their expression. Additionally, SlJMJ3 reduced the levels of H3K27me3 at its target genes, thereby up-regulating their expression. In summary, our findings highlight the role of SlJMJ3 in the regulation of fruit ripening in tomato. By removing the methyl group from trimethylated histone H3 lysine 27 at ripening-related genes, SlJMJ3 acts as an epigenetic regulator that orchestrates the complex molecular processes underlying fruit ripening.

Water permeability of different aerial tissues of carnations is related to cuticular wax composition.

Cuticular wax protects aerial plant tissues against uncontrolled water loss. To compare the differences among tissues, cultivars, and postharvest stages, we characterized the surface morphology, water permeability, and chemical composition of cuticular wax on the leaf, calyx, and petals of two carnation cultivars ('Master' and 'Lady green') at two postharvest stages. Obvious differences in these characteristics were found among tissues but not among cultivars or postharvest stages. The leaf surface was relatively smooth, whereas convex cells were observed on the petals. The mean minimum conductance of leaves was significantly higher than that of the calyx, followed by that of petals. It ranged between 8.8 × 10-4  m s-1 for 'Lady green' leaves at Stage II and 3.6 × 10-5  m s-1 for 'Master' petals at Stage I. Petal wax contained high concentrations of n-alkanes, whereas primary alcohols dominated in leaf wax. The weighted average chain length (ACL) was higher in petal wax than in leaf wax; it ranged from 19.6 in 'Lady green' leaves to 24.14 in 'Lady green' petals at Stage I. In conclusion, carnation petals are characterized by numerous convex cells on both the adaxial and abaxial surfaces, and their main cuticular wax components, alkanes, have a higher ACL than leaf cuticular wax, which contributes to their higher water barrier property. The results provide further evidence for the association between cuticular chemical composition and the physiological function of the cuticle in blocking water transpiration.

SlJMJ7 orchestrates tomato fruit ripening via crosstalk between H3K4me3 and DML2-mediated DNA demethylation.

The ripening of fleshy fruits is a unique developmental process that Arabidopsis and rice lack. This process is driven by hormones and transcription factors. However, the critical and early regulators of fruit ripening are still poorly understood. Here, we revealed that SlJMJ7, an H3K4 demethylase, is a critical negative regulator of fruit ripening in tomato. Combined genome-wide transcription, binding sites, histone H3K4me3 and DNA methylation analyses demonstrated that SlJMJ7 regulates a key group of ripening-related genes, including ethylene biosynthesis (ACS2, ACS4 and ACO6), transcriptional regulation (RIN and NOR) and DNA demethylation (DML2) genes, by H3K4me3 demethylation. Moreover, loss of SlJMJ7 function leads to increased H3K4me3 levels, which directly activates ripening-related genes, and to global DML2-mediated DNA hypomethylation in fruit, which indirectly prompts expression of ripening-related genes. Together, these effects lead to accelerated fruit ripening in sljmj7 mutant. Our findings demonstrate that SlJMJ7 acts as a master negative regulator of fruit ripening not only through direct removal of H3K4me3 from multiple key ripening-related factors, but also through crosstalk between histone and DNA demethylation. These findings reveal a novel crosstalk between histone methylation and DNA methylation to regulate gene expression in plant developmental processes.

Biosynthesis, regulation, and biological significance of fumonisins in fungi: current status and prospects.

Fumonisins are one of the most important mycotoxin classes due to their widespread occurrence and potential health threat to humans and animals. Currently, most of the research focuses on the control of fumonisin contamination in the food supply chain. In recent years, significant progress in biochemistry, enzymology, and genetic regulation of fumonisin biosynthesis has been achieved using molecular technology. Furthermore, new insights into the roles of fumonisins in the interaction between fungi and plant hosts have been reported. This review provides an overview of the current understanding of the biosynthesis and regulation of fumonisins. The ecological significance of fumonisins to Fusarium species that produce the toxins is discussed, and the complex regulatory networks of fumonisin synthesis is proposed.

Research progress on the application of different preservation methods for controlling fungi and toxins in fruit and vegetable.

Fruits and vegetables are susceptible to fungal infections during picking, transportation, storage and processing, which have a high potential to produce toxins. Fungi and toxins can cause acute or chronic poisoning after entering the body. In the field of fruit and vegetable preservation, technologies such as temperature control, modified atmosphere, irradiation, application of natural or chemical preservatives, and edible films are commonly used. In practical applications, according to the types, physiological differences and actual needs of fruits and vegetables, suitable preservation methods can be selected to achieve the effect of preservation and control of fungi and toxins. The starting point of fresh-keeping technology is to delay post-harvest senescence of fruits and vegetables, inhibit the respiratory intensity, and control the reproduction of microorganisms, which is important to control the reproduction of fungi and the production of toxins. From the three directions of physical, chemical and biological means, the article analyses and explores the effects of different external factors on the production of toxins and the effects of different preservation techniques on fungal growth and toxin production in fruits and vegetables, in order to provide new ideas for the preservation of fruits and vegetables and the control of harmful substances in food.

Arsenic causes mitochondrial biogenesis obstacles by inhibiting the AMPK/PGC-1α signaling pathway and also induces apoptosis and dysregulated mitophagy in the duck liver.

Arsenic is a dangerous metalloid-material which is known to cause liver injury in many animals and humans. However, little is known about the underlying mechanism of arsenic-induced hepatotoxicity in poultry. This study was executed to systematically investigate the potential role of mitochondrial biogenesis, mitophagy and apoptosis in duck hepatotoxicity caused by arsenic. Results showed that the body weight and liver coefficient of duck had distinct changed after arsenic-exposure, and the arsenic content in serum and liver also increased significantly in a dose-dependent manner. Meanwhile, histopathological examination and metabolomics results showed that arsenic-exposure caused severe steatosis and metabolism disorder in liver tissues. Furthermore, arsenic-exposure significantly inhibited AMPK/PGC-1α-mediated mitochondrial biogenesis, determined by the ultrastructure observation and down-regulation of p-AMPKα/AMPKα, PGC-1α, NRF1, NRF2, TFAM, TFB1M, TFB2M and COX-Ⅳ expression levels. Besides, arsenic-treatment obviously increased the levels of mitophagy (PINK1, Parkin, LC3, P62) and pro-apoptotic (Caspase-3, Caspase-9, Cleaved Caspase-3, Cytc, Bax, P53) indexes, and simultaneously resulted in reductions in anti-apoptosis index (Bcl-2). Overall, our findings provided evidences that arsenic-induced duck hepatotoxicity may be caused by a combination of impaired mitochondrial biosynthesis, mitophagy, and mitochondrial-dependent apoptosis. To our knowledge, this is the first report to systematically investigate the potential mechanism of arsenic-induced hepatotoxicity in poultry.

Effects of exogenous sucrose and selenium on plant growth, quality, and sugar metabolism of pea sprouts.

BACKGROUND: Pea sprouts are considered a healthy food. Sucrose is a key nutritional factor affecting taste and flavor. Meanwhile, selenium (Se) is an essential micronutrient that plays multiple roles in wide variety of physiological processes and improves crop quality and nutritional value. Nonetheless, the effects of the combination of sucrose and Se treatment on growth, quality, and sugar metabolism of pea sprouts have not been explored. RESULTS: The results revealed that sucrose at 10 mg L-1 obviously increased fresh weight, vitamin C, soluble protein, soluble sugar, fructose, glucose, and sucrose contents. Se treatments also improved nutritional quality, but higher Se (2.5 mg L-1 ) significantly inhibited the growth of seedlings. Interestingly, the combined application of sucrose (10 mg L-1 ) and Se (1.25 mg L-1 ) could effectively promote vitamin C, sucrose, and fructose contents, especially the Se content, compared with Se application alone. Additionally, there were significant differences in the regulation of sugar metabolism between Se alone and combined application of sucrose and Se. Acid invertase and neutral invertase play a pivotal role in the accumulation of soluble sugar under Se treatments alone, and acid invertase might be the key enzyme to limit sugar accumulation under combined application of sucrose and Se. CONCLUSION: The moderate combined application of sucrose (10 mg L-1 ) and Se (1.25 mg L-1 ) more effectively regulated sugar metabolism and improved nutritional quality than Se application alone did. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Advances in chilling injury of postharvest fruit and vegetable: Extracellular ATP aspects.

Due to the global use of cold chain, the development of postharvest technology to reduce chilling injury (CI) in postharvest fruits and vegetables during storage and transport is needed urgently. Considerable evidence shows that maintaining intracellular adenosine triphosphate (iATP) in harvested fruits and vegetables is beneficial to inhibiting CI occurrence. Extracellular ATP (eATP) is a damage-associated signal molecule and plays an important role in CI of postharvest fruits and vegetables through its receptor and subsequent signal transduction under low-temperature stress. The development of new aptasensors for the simultaneous determination of eATP level allows for better understanding of the roles of eATP in a myriad of responses mediated by low-temperature stress in relation to the chilling tolerance of postharvest fruits and vegetables. The multiple biological functions of eATP and its receptors in postharvest fruits and vegetables were attributed to interactions with reactive oxygen species (ROS) and nitric oxide (NO) in coordination with phytohormones and other signaling molecules via downstream physiological activities. The complicated interconnection among eATP in relation to its receptors, eATP/iATP homeostasis, ROS, NO, and heat shock proteins triggered by eATP recognition has been emphasized. This paper reviews recent advances in the beneficial effects of energy handling, outlines the production and homeostasis of eATP, discusses the possible mechanism of eATP and its receptors in chilling tolerance, and provides future research directions for CI in postharvest fruits and vegetables during low-temperature storage.

An ethylene-hypersensitive methionine sulfoxide reductase regulated by NAC transcription factors increases methionine pool size and ethylene production during kiwifruit ripening.

Ethylene plays an important role in regulating fruit ripening by triggering dynamic changes in expression of ripening-associated genes, but the functions of many of these genes are still unknown. Here, a methionine sulfoxide reductase gene (AdMsrB1) was identified by transcriptomics-based analysis as the gene most responsive to ethylene treatment in ripening kiwifruit. The AdMsrB1 protein exhibits a stereospecific activity toward the oxidative stress-induced R enantiomer of methionine sulfoxide (MetSO), reducing it to methionine (Met). Stable overexpression of AdMsrB1 in kiwifruit significantly increased the content of free Met and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and increased ethylene production. Dual-luciferase assays indicated that the AdMsrB1 promoter was not directly upregulated by ethylene treatment but was modulated by two ethylene-inducible NAM/ATAF/CUC transcription factors (AdNAC2 and AdNAC72) that bind directly to the AdMsrB1 promoter. Overexpression of AdNAC72 in kiwifruit not only enhanced AdMsrB1 expression, but also increased free Met and ACC content and ethylene production rates. This finding establishes an unexpected regulatory loop that enhances ethylene production and the concentration of its biosynthetic intermediates.

Redox Regulation of the NOR Transcription Factor Is Involved in the Regulation of Fruit Ripening in Tomato.

Transcription factors (TFs) are important regulators of plant growth and development and responses to stresses. TFs themselves are also prone to multiple posttranslational modifications (PTMs). However, redox-mediated PTM of TFs in plants remains poorly understood. Here, we established that NON-RIPENING (NOR), a master TF regulating tomato (Solanum lycopersicum) fruit ripening, is a target of the Met sulfoxide reductases A and B, namely E4 and SlMsrB2, respectively, in tomato. Met oxidation in NOR, i.e. sulfoxidation, or mimicking sulfoxidation by mutating Met-138 to Gln, reduces its DNA-binding capacity and transcriptional regulatory activity in vitro. E4 and SlMsrB2 partially repair oxidized NOR and restore its DNA-binding capacity. Transgenic complementation of the nor mutant with NOR partially rescues the ripening defects. However, transformation of nor with NOR-M138Q, containing mimicked Met sulfoxidation, inhibits restoration of the fruit ripening phenotype, and this is associated with the decreased DNA-binding and transcriptional activation of a number of ripening-related genes. Taken together, these observations reveal a PTM mechanism by which Msr-mediated redox modification of NOR regulates the expression of ripening-related genes, thereby influencing tomato fruit ripening. Our report describes how sulfoxidation of TFs regulates developmental processes in plants.

Sulfoxidation regulation of transcription factor NAC42 influences its functions in relation to stress-induced fruit ripening in banana.

Redox modification of functional or regulatory proteins has emerged as an important mechanism of post-translational modification. However, the role of redox modifications of transcription factors mediated by methionine sulfoxide reductase (Msr) in regulating physiological processes in plants remains unclear, especially in fruit ripening. In this study, we determined that MaNAC42, a transcriptional activator, is involved in the regulation of fruit ripening in banana under oxidative stress. Integrated analysis of ChIP-qPCR and EMSA data showed that MaNAC42 directly binds to promoters of genes related to oxidative stress and ripening. Ectopic overexpression of MaNAC42 in Arabidopsis delays dark-induced senescence in leaves, indicating that MaNAC42 plays a negative role in senescence. Furthermore, we found that MaNAC42 is a target of MaMsrB2, a methionine sulfoxide reductase B. Methionine oxidation in MaNAC42 (i.e. sulfoxidation) or mimicking sulfoxidation by mutating methionine to glutamine both lead to decreased DNA-binding capacity and transcriptional activity. On the other hand, MaMsrB2 can partially repair oxidized MaNAC42 and restore its DNA-binding capacity. Thus, our results suggest a novel regulatory mechanism of fruit ripening in banana involving MaMsrB2-mediated redox regulation of the ripening-related transcription factor MaNAC42.

Alternative splicing of MaMYB16L regulates starch degradation in banana fruit during ripening.

The alternative splicing of select genes is an important mechanism to regulate responses to endogenous and environmental signals in plants. However, the role of alternative splicing in regulating fruit ripening remains unclear. Here, we discovered that MaMYB16L, an R1-type MYB transcription factor, undergoes alternative splicing and generates two transcripts, the full-length isoform MaMYB16L and a truncated form MaMYB16S, in banana fruit. During banana fruit ripening, the alternative splicing process intensifies with downregulated MaMYB16L and upregulated MaMYB16S. Moreover, MaMYB16L is a transcriptional repressor that directly binds with the promoters of many genes associated with starch degradation and MaDREB2, a positive ripening regulator, and represses their expression. In contrast, MaMBY16S lacks a DNA-binding domain but competitively combines and forms non-functional heterodimers with functional MaMYB16L. MaMYB16L-MaMYB16S heterodimers decrease the binding capacity and transrepression activity of MaMYB16L. The downregulation of MaMYB16L and the upregulation of MaMYB16S, that is, a decreased ratio of active to non-active isoforms, facilitates the activation of ripening-related genes and thereby promotes fruit ripening. Furthermore, the transient overexpression of MaMYB16S promotes banana fruit ripening, whereas the overexpression of MaMYB16L delays this process. Therefore, the alternative splicing of MaMYB16L might generate a self-controlled regulatory loop to regulate banana fruit ripening.

MicroRNA528, a hub regulator modulating ROS homeostasis via targeting of a diverse set of genes encoding copper-containing proteins in monocots.

Plant microRNAs (miRNAs) regulate vital cellular processes, including responses to extreme temperatures with which reactive oxygen species (ROS) are often closely associated. In the present study, it was found that aberrant temperatures caused extensive changes in abundance to numerous miRNAs in banana fruit, especially the copper (Cu)-associated miRNAs. Among them, miR528 was significantly downregulated under cold stress and it was found to target genes encoding polyphenol oxidase (PPO), different from those identified in rice and maize. Expression of PPO genes was upregulated by > 100-fold in cold conditions, leading to ROS surge and subsequent peel browning of banana fruit. Extensive comparative genomic analyses revealed that the monocot-specific miR528 can potentially target a large collection of genes encoding Cu-containing proteins. Most of them are actively involved in cellular ROS metabolism, including not only ROS generating oxidases, but also ROS scavenging enzymes. It also was demonstrated that miR528 has evolved a distinct preference of target genes in different monocots, with its target site varying in position among/within gene families, implying a highly dynamic process of target gene diversification. Its broad capacity to target genes encoding Cu-containing protein implicates miR528 as a key regulator for modulating the cellular ROS homeostasis in monocots.

Histone demethylase SlJMJ6 promotes fruit ripening by removing H3K27 methylation of ripening-related genes in tomato.

Fruit ripening is governed by a complex regulatory network. Reversible histone methylation and demethylation regulate chromatin structure and gene expression. However, little is known about the involvement of histone demethylases in regulating fruit ripening. Here, we found that the tomato (Solanum lycopersicum) SlJMJ6 encodes a histone lysine demethylase that specifically demethylates H3K27 methylation. Overexpression of SlJMJ6 accelerates tomato fruit ripening, which is associated with the upregulated expression of a large number of ripening-related genes. Integrated analysis of RNA-seq and chromatin immunoprecipitation followed by sequencing identified 32 genes directly targeted by SlJMJ6 and transcriptionally upregulated with decreased H3K27m3 in SlJMJ6-overexpressed fruit. Numerous SlJMJ6-regulated genes are involved in transcription regulation, ethylene biosynthesis, cell wall degradation and hormone signaling. Eleven ripening-related genes including RIPENING INHIBITOR (RIN), 1-aminocyclopropane 1-carboxylate synthase-4 (ACS4), 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1), pectate lyase (PL) and beta-galactosidase 4 (TBG4), and a DNA demethylase DML2, were confirmed to be regulated directly by SlJMJ6 through removing H3K27me3. Our results demonstrate that SlJMJ6 is a ripening-prompting H3K27me3 demethylase that activates the expression of the ripening-related genes by modulating H3K27me3, thereby facilitating tomato fruit ripening. Our work also reveals a novel link between histone demethylation and DNA demethylation in regulating fruit ripening. To our knowledge, this is the first report of the involvement of a histone lysine demethylase in the regulation of fruit ripening.

Banana sRNAome and degradome identify microRNAs functioning in differential responses to temperature stress.

BACKGROUND: Temperature stress is a major environmental factor affecting not only plant growth and development, but also fruit postharvest life and quality. MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in various biological processes. Harvested banana fruit can exhibit distinct symptoms in response to different temperature stresses, but the underlying miRNA-mediated regulatory mechanisms remained unknown. RESULTS: Here, we profiled temperature-responsive miRNAs in banana, using deep sequencing and computational and molecular analyses. In total 113 known miRNAs and 26 novel banana-specific miRNAs were identified. Of these miRNAs, 42 miRNAs were expressed differentially under cold and heat stresses. Degradome sequencing identified 60 target genes regulated by known miRNAs and half of these targets were regulated by 15 temperature-responsive miRNAs. The correlative expression patterns between several miRNAs and their target genes were further validated via qRT-PCR. Our data showed that miR535 and miR156 families may derive from a common ancestor during evolution and jointly play a role in fine-tuning SPL gene expression in banana. We also identified the miRNA-triggered phased secondary siRNAs in banana and found miR393-TIR1/AFB phasiRNA production displaying cold stress-specific enrichment. CONCLUSIONS: Our results provide a foundation for understanding the miRNA-dependent temperature stress response in banana. The characterized correlations between miRNAs and their response to temperature stress could serve as markers in the breeding programs or tools for improving temperature tolerance of banana.

Proteomic and transcriptomic analysis to unravel the influence of high temperature on banana fruit during postharvest storage.

Banana, an important food, incurs significant economic losses due to high storage temperature. Integrative analysis of proteome and transcriptome profiles of the banana peel stored at 20 °C (control) and 30 °C (HT) was used to investigate the molecular mechanism in response to high temperature stress. Critical proteins and genes relating to the response of banana fruit to HT stress were evaluated using partial least squares-discriminant analysis (PLS-DA) and orthogonal signal correction partial least squares-discriminant analysis (OPLS-DA). HT stress influenced proteins/genes related to chlorophyll metabolism, fruit firmness, signal transduction, energy metabolism, and stress response and defense. Together with scanning electron microscopy (SEM) and real time quantitative PCR (RT-qPCR) results, it can be concluded that HT stress resulted in stay-green ripening of banana fruit. Additionally, HT stress accelerated firmness loss and senescence of banana peel, might mainly through regulating hormone signaling pathway, stress protective ability, and energy metabolism in the banana peel. Our study provided a clearer understanding of regulatory mechanisms of HT treatment on banana fruit and potential genetic resources for the improvement of high temperature-tolerant characteristics in banana fruit.

Identification of HDA15-PIF1 as a key repression module directing the transcriptional network of seed germination in the dark.

Light is a major external factor in regulating seed germination. Photoreceptor phytochrome B (PHYB) plays a predominant role in promoting seed germination in the initial phase after imbibition, partially by repressing phytochrome-interacting factor1 (PIF1). However, the mechanism underlying the PHYB-PIF1-mediated transcription regulation remains largely unclear. Here, we identified that histone deacetylase15 (HDA15) is a negative component of PHYB-dependent seed germination. Overexpression of HDA15 in Arabidopsis inhibits PHYB-dependent seed germination, whereas loss of function of HDA15 increases PHYB-dependent seed germination. Genetic evidence indicated that HDA15 acts downstream of PHYB and represses seed germination dependent on PIF1. Furthermore, HDA15 interacts with PIF1 both in vitro and in vivo. Genome-wide transcriptome analysis revealed that HDA15 and PIF1 co-regulate the transcription of the light-responsive genes involved in multiple hormonal signaling pathways and cellular processes in germinating seeds in the dark. In addition, PIF1 recruits HDA15 to the promoter regions of target genes and represses their expression by decreasing the histone H3 acetylation levels in the dark. Taken together, our analysis uncovered the role of histone deacetylation in the light-regulated seed germination process and identified that HDA15-PIF1 acts as a key repression module directing the transcription network of seed germination.

Label-Free Proteomic Analysis of Molecular Effects of 2-Methoxy-1,4-naphthoquinone on Penicillium italicum.

Penicillium italicum is the principal pathogen causing blue mold of citrus. Searching for novel antifungal agents is an important aspect of the post-harvest citrus industry because of the lack of higher effective and low toxic antifungal agents. Herein, the effects of 2-methoxy-1,4-naphthoquinone (MNQ) on P. italicum and its mechanism were carried out by a series of methods. MNQ had a significant anti-P. italicum effect with an MIC value of 5.0 µg/mL. The label-free protein profiling under different MNQ conditions identified a total of 3037 proteins in the control group and the treatment group. Among them, there were 129 differentially expressed proteins (DEPs, up-regulated > 2.0-fold or down-regulated < 0.5-fold, p < 0.05), 19 up-regulated proteins, 26 down-regulated proteins, and 67 proteins that were specific for the treatment group and another 17 proteins that were specific for the control group. Of these, 83 proteins were sub-categorized into 23 hierarchically-structured GO classifications. Most of the identified DEPs were involved in molecular function (47%), meanwhile 27% DEPs were involved in the cellular component and 26% DEPs were involved in the biological process. Twenty-eight proteins identified for differential metabolic pathways by KEGG were sub-categorized into 60 classifications. Functional characterization by GO and KEGG enrichment results suggests that the DEPs are mainly related to energy generation (mitochondrial carrier protein, glycoside hydrolase, acyl-CoA dehydrogenase, and ribulose-phosphate 3-epimerase), NADPH supply (enolase, pyruvate carboxylase), oxidative stress (catalase, glutathione synthetase), and pentose phosphate pathway (ribulose-phosphate 3-epimerase and xylulose 5-phosphate). Three of the down-regulated proteins selected randomly the nitro-reductase family protein, mono-oxygenase, and cytochrome P450 were verified using parallel reaction monitoring. These findings illustrated that MNQ may inhibit P. italicum by disrupting the metabolic processes, especially in energy metabolism and stimulus response that are both critical for the growth of the fungus. In conclusion, based on the molecular mechanisms, MNQ can be developed as a potential anti-fungi agent against P. italicum.

Sulfoxidation Regulation of Musa acuminata Calmodulin (MaCaM) Influences the Functions of MaCaM-Binding Proteins.

Sulfoxidation of methionine in proteins by reactive oxygen species can cause conformational alteration or functional impairment, and can be reversed by methionine sulfoxide reductase (Msr). Currently, only a few potential Msr substrates have been confirmed in higher plants. Here, we investigated Msr-mediated sulfoxidation regulation of calmodulin (CaM) and its underlying biological significance in relation to banana fruit ripening and senescence. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. We verified that MaCaM1 interacts with MaMsrA7 in vitro and in vivo, and sulfoxidated MaCaM1 could be partly repaired by MaMsrA7 (MaMsrA7 reduces oxidized residues Met77 and Met110 in MaCaM1). Furthermore, we investigated two known CaM-binding proteins, catalase (MaCAT1) and MaHY5-1. MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3) in banana fruit. MaCaM1 could enhance the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1 toward MaPSY2. Mimicked sulfoxidation in MaCaM1 did not affect the physical interactions of the protein with MaHY5-1 and MaCAT1, but reduced the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1. Our data suggest that sulfoxidation modification in MaCaM1 by MaMsrA7 regulates antioxidant response and gene transcription, thereby being involved in regulation of ripening and senescence of banana fruit.