mucG, mucH, and mucI Modulate Production of Mutanocyclin and Reutericyclins in Streptococcus mutans B04Sm5.

Streptococcus mutans is considered a primary etiologic agent of dental caries, which is the most common chronic infectious disease worldwide. S. mutans B04Sm5 was recently shown to produce reutericyclins and mutanocyclin through the muc biosynthetic gene cluster and to utilize reutericyclins to inhibit the growth of neighboring commensal streptococci. In this study, examination of S. mutans and muc phylogeny suggested evolution of an ancestral S. mutans muc into three lineages within one S. mutans clade and then horizontal transfer of muc to other S. mutans clades. The roles of the mucG and mucH transcriptional regulators and the mucI transporter were also examined. mucH was demonstrated to encode a transcriptional activator of muc. mucH deletion reduced production of mutanocyclin and reutericyclins and eliminated the impaired growth and inhibition of neighboring streptococci phenotypes, which are associated with reutericyclin production. ΔmucG had increased mutanocyclin and reutericyclin production, which impaired growth and increased the ability to inhibit neighboring streptococci. However, deletion of mucG also caused reduced expression of mucD, mucE, and mucI. Deletion of mucI reduced mutanocyclin and reutericylin production but enhanced growth, suggesting that mucI may not transport reutericyclin as its homolog does in Limosilactobacillus reuteri. Further research is needed to determine the roles of mucG and mucI and to identify any cofactors affecting the activity of the mucG and mucH regulators. Overall, this study provided pangenome and phylogenetic analyses that serve as a resource for S. mutans research and began elucidation of the regulation of reutericyclins and mutanocyclin production in S. mutans. IMPORTANCE S. mutans must be able to outcompete neighboring organisms in its ecological niche in order to cause dental caries. S. mutans B04Sm5 inhibited the growth of neighboring commensal streptococci through production of reutericyclins via the muc biosynthetic gene cluster. In this study, an S. mutans pangenome database and updated phylogenetic tree were generated that will serve as valuable resources for the S. mutans research community and that provide insights into the carriage and evolution of S. mutans muc. The MucG and MucH regulators, and the MucI transporter, were shown to modulate production of reutericyclins and mutanocyclin. These genes also affected the ability of S. mutans to inhibit neighboring commensals, suggesting that they may play a role in S. mutans virulence.

(S)-Reutericyclin: Susceptibility Testing and In Vivo Effect on Murine Fecal Microbiome and Volatile Organic Compounds.

We aimed to assess the in vitro antimicrobial activity and the in vivo effect on the murine fecal microbiome and volatile organic compound (VOC) profile of (S)-reutericyclin. The antimicrobial activity of (S)-reutericyclin was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Staphylococcus (S.) epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa and Propionibacterium acnes. Reutericyclin or water were gavage fed to male BALBc mice for 7 weeks. Thereafter stool samples underwent 16S based microbiome analysis and VOC analysis by gas chromatography mass spectrometry (GC-MS). (S)-reutericyclin inhibited growth of S. epidermidis only. Oral (S)-reutericyclin treatment caused a trend towards reduced alpha diversity. Beta diversity was significantly influenced by reutericyclin. Linear discriminant analysis Effect Size (LEfSe) analysis showed an increase of Streptococcus and Muribaculum as well as a decrease of butyrate producing Ruminoclostridium, Roseburia and Eubacterium in the reutericyclin group. VOC analysis revealed significant increases of pentane and heptane and decreases of 2,3-butanedione and 2-heptanone in reutericyclin animals. The antimicrobial activity of (S)-reutericyclin differs from reports of (R)-reutericyclin with inhibitory effects on a multitude of Gram-positive bacteria reported in the literature. In vivo (S)-reutericyclin treatment led to a microbiome shift towards dysbiosis and distinct alterations of the fecal VOC profile.

Effect of copy number of the spoVA2mob operon, sourdough and reutericyclin on ropy bread spoilage caused by Bacillus spp.

Bacillus spp. cause ropy bread spoilage of bread, which is characterized by a rotten fruity odor, followed by discoloration and degradation of the crumb. Bacillus spp. are wheat grain endophytes and form heat resistant endospores, therefore, process hygiene and heating during baking do not prevent ropy spoilage. This study used 8 strains of Bacillus subtilis and Bacillus amyloliquefaciens to determine whether the presence and the copy number of spoVA2mob operon influences survival after baking; in addition, the spoilage phenotype was correlated with the presence of amylolytic enzymes in genomes of Bacillus spp.. The presence and copy number of the spoVA2mob operon had only a minor effect on survival of Bacillus endospores. Strains of B. amyloliquefaciens caused ropy spoilage faster than strains of B. subtilis, this difference correlated to the number and type of extracellular amylases encoded in the genomes of the strains of B. amyloliquefaciens and B. subtilis. The inhibitory effect of sourdough on ropy spoilage was determined by addition of 3-24% sourdough fermented with L. reuteri TMW1.656. Addition of 12% and 24% sourdough, corresponding to a bread pH of 5.93 ± 0.041 and 5.53 ± 0.040, respectively, delayed ropy spoilage for 2 and more than 5 d, respectively. The comparison of addition of 12% sourdough fermented with the reutericyclin producing L. reuteri TMW1.656 and the isogenic reutericyclin-negative strain L. reuteri TMW1.656ΔgtfAΔrtcN demonstrated that reutericyclin produced in sourdough inhibits growth of Bacillus in bread. In conclusion, sourdough inhibits germination of Bacillus spores in bread and the effect of sourdough is enhanced by reutericyclin.

Glycerol Monolaurate, an Analogue to a Factor Secreted by Lactobacillus, Is Virucidal against Enveloped Viruses, Including HIV-1.

The vaginal microbiota influences sexual transmission of human immunodeficiency virus type 1 (HIV-1). Colonization of the vaginal tract is normally dominated by Lactobacillus species. Both Lactobacillus and Enterococcus faecalis may secrete reutericyclin, which inhibits the growth of a variety of pathogenic bacteria. Increasing evidence suggests a potential therapeutic role for an analogue of reutericyclin, glycerol monolaurate (GML), against microbial pathogens. Previous studies using a macaque vaginal simian immunodeficiency virus (SIV) transmission model demonstrated that GML reduces transmission and alters immune responses to infection in vitro Previous studies showed that structural analogues of GML negatively impact other enveloped viruses. We sought to expand understanding of how GML inhibits HIV-1 and other enveloped viruses and show that GML restricts HIV-1 entry post-CD4 engagement at the step of coreceptor binding. Further, HIV-1 and yellow fever virus (YFV) particles were more sensitive to GML interference than particles "matured" by proteolytic processing. We show that high-pressure-liquid-chromatography (HPLC)-purified reutericyclin and reutericyclin secreted by Lactobacillus inhibit HIV-1. These data emphasize the importance and protective nature of the normal vaginal flora during viral infections and provide insights into the antiviral mechanism of GML during HIV-1 infection and, more broadly, to other enveloped viruses.IMPORTANCE A total of 340 million sexually transmitted infections (STIs) are acquired each year. Antimicrobial agents that target multiple infectious pathogens are ideal candidates to reduce the number of newly acquired STIs. The antimicrobial and immunoregulatory properties of GML make it an excellent candidate to fit this critical need. Previous studies established the safety profile and antibacterial activity of GML against both Gram-positive and Gram-negative bacteria. GML protected against high-dose SIV infection and reduced inflammation, which can exacerbate disease, during infection. We found that GML inhibits HIV-1 and other human-pathogenic viruses (yellow fever virus, mumps virus, and Zika virus), broadening its antimicrobial range. Because GML targets diverse infectious pathogens, GML may be an effective agent against the broad range of sexually transmitted pathogens. Further, our data show that reutericyclin, a GML analog expressed by some lactobacillus species, also inhibits HIV-1 replication and thus may contribute to the protective effect of Lactobacillus in HIV-1 transmission.

Cariogenic Streptococcus mutans Produces Tetramic Acid Strain-Specific Antibiotics That Impair Commensal Colonization.

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

Glycerol Monolaurate Contributes to the Antimicrobial and Anti-inflammatory Activity of Human Milk.

Human milk has antimicrobial compounds and immunomodulatory activities. We investigated glycerol monolaurate (GML) in human milk versus bovine milk and infant formula for antimicrobial and anti-inflammatory activities. Human milk contained approximately 3000 µg/ml of GML, compared to 150 µg/ml in bovine milk and none in infant formula. For bacteria tested (Staphylococcus aureus, Bacillus subtilis, Clostridium perfringens, Escherichia coli), except Enterococcus faecalis, human milk was more antimicrobial than bovine milk and formula. The Enterococcus faecalis strain, which was not inhibited, produced reutericyclin, which is an analogue of GML and functions as a growth stimulant in bacteria that produce it. Removal of GML and other lipophilic molecules from human milk by ethanol extraction resulted in a loss of antibacterial activity, which was restored by re-addition of GML. GML addition caused bovine milk to become antimicrobial. Human milk but not bovine milk or formula inhibited superantigen and bacterial-induced IL-8 production by model human epithelial cells. GML may contribute beneficially to human milk compared to bovine milk or infant formula.

Aminotenuazonic Acid: Isolation, Structure Elucidation, Total Synthesis and Herbicidal Activity of a New Tetramic Acid from Fruiting Bodies of Laccaria Species.

(5S,6S)-Aminotenuazonic acid, a new 3-acyltetramic acid, related to the well-known mycotoxin tenuazonic acid has been isolated from fruiting bodies of Laccaria bicolor. Its structure was mostly established by analysis of its 2D NMR and HR-(+)-ESI-MS spectra. A total synthesis starting from N-Boc-l-isoleucine gave (5S,6S)-aminotenuazonic acid in 8 % yield over nine steps (67 % de). The key steps of the total synthesis are a light-initiated Hofmann-Löffler-Freytag radical chain reaction and a Dieckmann cyclisation. The relative and absolute configurations of the natural product were determined by comparison of its NMR and CD spectra with those of the corresponding enantiopure synthetic compounds. Metabolic profiling of crude extracts of different mushrooms showed that aminotenuazonic acid is present in all four of the investigated Laccaria species. Aminotenuazonic acid shows phytotoxic activities against the root and shoot growth of Lepidium sativum, Pinus sylvestris and Arabidopsis thaliana comparable to those of tenuazonic acid.

A Synthetic Route to ß-Hydroxytyrosine-Derived Tetramic Acids: Total Synthesis of the Fungal Metabolite F-14329.

3-Acyltetramic acids derived from ß-hydroxytyrosine are synthetically challenging. The first route to this structural motif, based upon a condensation between a Meldrum's acid conjugate bearing the acyl side chain, and a ß-hydroxytyrosinate, N-protected by an ortho-nitrobenzyl group is presented. This group enables the Dieckmann cyclization of the resulting N-(ß-ketoacyl)amino ester, after which it can be removed photolytically without compromising the delicate 3'-hydroxy group. This strategy was applied to the first total synthesis of the fungal metabolite F-14329 (1).

Genome mining unveils widespread natural product biosynthetic capacity in human oral microbe Streptococcus mutans.

Streptococcus mutans is a major pathogen causing human dental caries. As a Gram-positive bacterium with a small genome (about 2 Mb) it is considered a poor source of natural products. Due to a recent explosion in genomic data available for S. mutans strains, we were motivated to explore the natural product production potential of this organism. Bioinformatic characterization of 169 publically available genomes of S. mutans from human dental caries revealed a surprisingly rich source of natural product biosynthetic gene clusters. Anti-SMASH analysis identified one nonribosomal peptide synthetase (NRPS) gene cluster, seven polyketide synthase (PKS) gene clusters and 136 hybrid PKS/NRPS gene clusters. In addition, 211 ribosomally synthesized and post-translationally modified peptides (RiPPs) clusters and 615 bacteriocin precursors were identified by a combined analysis using BAGEL and anti-SMASH. S. mutans harbors a rich and diverse natural product genetic capacity, which underscores the importance of probing the human microbiome and revisiting species that have traditionally been overlooked as "poor" sources of natural products.

Equisetin, reutericyclin and streptolodygin as natural product lead structures for novel antibiotic libraries.

The emergence of antimicrobial resistance has created a need for the development of novel antibacterial therapies to treat infection. Natural products that exhibit antibacterial activity offer validated starting points for library generation, and the authors report here that small molecule mimics of tetramate-containing natural products may show antibacterial activity and offer the potential for further optimization.

Gastrointestinal localization of metronidazole by a lactobacilli-inspired tetramic acid motif improves treatment outcomes in the hamster model of Clostridium difficile infection.

OBJECTIVES: Metronidazole, a mainstay treatment for Clostridium difficile infection (CDI), is often ineffective for severe CDI. Whilst this is thought to arise from suboptimal levels of metronidazole in the colon due to rapid absorption, empirical validation is lacking. In contrast, reutericyclin, an antibacterial tetramic acid from Lactobacillus reuteri, concentrates in the gastrointestinal tract. In this study, we modified metronidazole with reutericyclin's tetramic acid motif to obtain non-absorbed compounds, enabling assessment of the impact of pharmacokinetics on treatment outcomes. METHODS: A series of metronidazole-bearing tetramic acid substituents were synthesized and evaluated in terms of anti-C. difficile activities, gastric permeability, in vivo pharmacokinetics, efficacy in the hamster model of CDI and mode of action. RESULTS: Most compounds were absorbed less than metronidazole in cell-based Caco-2 permeability assays. In hamsters, lead compounds compartmentalized in the colon rather than the bloodstream with negligible levels detected in the blood, in direct contrast with metronidazole, which was rapidly absorbed into the blood and was undetectable in caecum. Accordingly, four leads were more efficacious (P < 0.05) than metronidazole in C. difficile-infected animals. Improved efficacy was not due to an alternative mode of action, as the leads retained the mode of action of metronidazole. CONCLUSIONS: This study provides the clearest empirical evidence that the high absorption of metronidazole lowers treatment outcomes for CDI and suggests a role for the tetramic acid motif for colon-specific drug delivery. This approach also has the potential to lower systemic toxicity and drug interactions of nitroheterocyclic drugs for treating gastrointestine-specific diseases.

Genetic determinants of reutericyclin biosynthesis in Lactobacillus reuteri.

Reutericyclin is a unique antimicrobial tetramic acid produced by some strains of Lactobacillus reuteri. This study aimed to identify the genetic determinants of reutericyclin biosynthesis. Comparisons of the genomes of reutericyclin-producing L. reuteri strains with those of non-reutericyclin-producing strains identified a genomic island of 14 open reading frames (ORFs) including genes coding for a nonribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), homologues of PhlA, PhlB, and PhlC, and putative transport and regulatory proteins. The protein encoded by rtcN is composed of a condensation domain, an adenylation domain likely specific for d-leucine, and a thiolation domain. rtcK codes for a PKS that is composed of a ketosynthase domain, an acyl-carrier protein domain, and a thioesterase domain. The products of rtcA, rtcB, and rtcC are homologous to the diacetylphloroglucinol-biosynthetic proteins PhlABC and may acetylate the tetramic acid moiety produced by RtcN and RtcK, forming reutericyclin. Deletion of rtcN or rtcABC in L. reuteri TMW1.656 abrogated reutericyclin production but did not affect resistance to reutericyclin. Genes coding for transport and regulatory proteins could be deleted only in the reutericyclin-negative L. reuteri strain TMW1.656ΔrtcN, and these deletions eliminated reutericyclin resistance. The genomic analyses suggest that the reutericyclin genomic island was horizontally acquired from an unknown source during a unique event. The combination of PhlABC homologues with both an NRPS and a PKS has also been identified in the lactic acid bacteria Streptococcus mutans and Lactobacillus plantarum, suggesting that the genes in these organisms and those in L. reuteri share an evolutionary origin.

Chemical modulation of the biological activity of reutericyclin: a membrane-active antibiotic from Lactobacillus reuteri.

Whilst the development of membrane-active antibiotics is now an attractive therapeutic concept, progress in this area is disadvantaged by poor knowledge of the structure-activity relationship (SAR) required for optimizing molecules to selectively target bacteria. This prompted us to explore the SAR of the Lactobacillus reuteri membrane-active antibiotic reutericyclin, modifying three key positions about its tetramic acid core. The SAR revealed that lipophilic analogs were generally more active against Gram-positive pathogens, but introduction of polar and charged substituents diminished their activity. This was confirmed by cytometric assays showing that inactive compounds failed to dissipate the membrane potential. Radiolabeled substrate assays indicated that dissipation of the membrane potential by active reutericyclins correlated with inhibition of macromolecular synthesis in cells. However, compounds with good antibacterial activities also showed cytotoxicity against Vero cells and hemolytic activity. Although this study highlights the challenge of optimizing membrane-active antibiotics, it shows that by increasing antibacterial potency the selectivity index could be widened, allowing use of lower non-cytotoxic doses.

New routes towards reutericyclin analogues.

A range of N-acylpyrrolo[3,4-c]isoxazoles and derived N-acyltetramides has been prepared via a nitrile oxide dipolar cycloaddition approach, as analogues of the acyltetramic acid metabolite reutericyclin, of interest for its antibiotic potential against Gram-positive bacteria including hospital-acquired infections of resistant Clostridium difficile.

Development of analytical methods for the determination of tenuazonic acid analogues in food commodities.

Analogues of the Alternaria mycotoxin tenuazonic acid (TA, biosynthesized by the fungus from the amino acid isoleucine) derived from valine (ValTA), leucine (LeuTA), alanine (AlaTA) and phenylalanine (PheTA) were synthesized and characterized by mass spectrometry (MS) and (1)H nuclear magnetic resonance (NMR) spectroscopy. Concentrations of stock solutions were determined by quantitative (1)H NMR (qHNMR). Two analytical methods based on high performance liquid chromatography (HPLC) and MS detection were developed, one with derivatization with 2,4-dinitrophenylhydrazine (DNPH) and one without derivatization. Limits of detection (LODs) were between 1-3 µg/kg (with derivatization) and 50-80 µg/kg (without derivatization). Respective limits of quantitation (LOQs) were about three times higher. Beside TA, the analogues LeuTA (about 4% of TA content) and ValTA (about 10% of TA content) were found in highly contaminated sorghum infant cereals and sorghum grains. Other analogues were not detected. Quantification of LeuTA and ValTA was performed using [(13)C6,(15)N]-TA as internal standard and matrix matched calibration. Recovery was between 95±11% and 102±10% for both compounds. Precision (relative standard deviation of triplicate sorghum cereal analyses three times during 3 weeks) was 7% for TA (912±60 µg/kg), 17% for LeuTA (43±8 µg/kg) and 19% for ValTA (118±22 µg/kg). These results indicate that several TA-like compounds, which are not yet characterized in aspects of their toxic properties, were detected in sorghum based infant food highly contaminated with TA, already.

Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure.

The effects of high pressure, temperature, and antimicrobial compounds on endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L(-1) nisin or 6.4 mg L(-1) reutericyclin for 0-60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4',6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to endospores, with MIC values 0.23 ± 0.084 mg L(-1) and 1.11 ± 0.48 mg L(-1), respectively. Nisin increased DPA release when endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of endospores of Clostridium spp. and may have application in foods.

In situ determination of Clostridium endospore membrane fluidity during pressure-assisted thermal processing in combination with nisin or reutericyclin.

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.

The membrane as a target for controlling hypervirulent Clostridium difficile infections.

OBJECTIVES: The stationary phase of Clostridium difficile, which is primarily responsible for diarrhoeal symptoms, is refractory to antibiotic killing. We investigated whether disrupting the functions of the clostridial membrane is an approach to control C. difficile infections by promptly removing growing and non-growing cells. METHODS: The bactericidal activities of various membrane-active agents were determined against C. difficile logarithmic-phase and stationary-phase cultures and compared with known antibiotics. Their effects on the synthesis of ATP, toxins A/B and sporulation were also determined. The effect of rodent caecal contents on anti-difficile activities was examined using two reutericyclin lead compounds, clofazimine, daptomycin and other comparator antibiotics. RESULTS: Most membrane-active agents and partially daptomycin showed concentration-dependent killing of both logarithmic-phase and stationary-phase cultures. The exposure of cells to compounds at their MBC resulted in a rapid loss of viability with concomitant reductions in cellular ATP, toxins A/B and spore numbers. With the exception of nisin, these effects were not due to membrane pore formation. Interestingly, the activity of the proton ionophore nigericin significantly increased as the growth of C. difficile decreased, suggesting the importance of the proton gradient to the survival of non-growing cells. The activities of the lipophilic antimicrobials reutericyclins and clofazimine were reduced by caecal contents. CONCLUSIONS: These findings indicate that C. difficile is uniquely susceptible to killing by molecules affecting its membrane function and bioenergetics, indicating that the clostridial membrane is a novel antimicrobial target for agents to alleviate the burden of C. difficile infections.

Reactive oxygen species from chloroplasts contribute to 3-acetyl-5-isopropyltetramic acid-induced leaf necrosis of Arabidopsis thaliana.

3-Acetyl-5-isopropyltetramic acid (3-AIPTA), a derivate of tetramic acid, is responsible for brown leaf-spot disease in many plants and often kills seedlings of both mono- and dicotyledonous plants. To further elucidate the mode of action of 3-AIPTA, during 3-AIPTA-induced cell necrosis, a series of experiments were performed to assess the role of reactive oxygen species (ROS) in this process. When Arabidopsis thaliana leaves were incubated with 3-AIPTA, photosystem II (PSII) electron transport beyond Q(A) (the primary plastoquinone acceptor of PSII) and the reduction of the end acceptors at the PSI acceptor side were inhibited; this was followed by increase in charge recombination and electron leakage to O(2), resulting in chloroplast-derived oxidative burst. Furthermore, the main antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) lost their activity. Excess ROS molecules directly attacked a variety of cellular components and subsequently caused electrolyte leakage, lipid peroxidation and cell membrane disruption. Finally, this led to cell destruction and leaf tissue necrosis. Thus, 3-AIPTA-triggered leaf necrosis of Arabidopsis was found to be a result of direct oxidative injury from the chloroplast-originated ROS burst initiated by the inhibition of normal photosynthetic electron transport.