Action of the fungal compound citrinin, a bioherbicide candidate, on photosystem II.

BACKGROUND: Bioherbicides are becoming more attractive as safe weed control tools towards sustainable agriculture. Natural products constitute an important source chemicals and chemical leads for discovery and development of novel pesticide target sites. Citrinin is a bioactive compound produced by fungi of the genera Penicillium and Aspergillus. However, its physiological-biochemical mechanism as a phytotoxin remains unclear. RESULTS: Citrinin causes visible leaf lesions on Ageratina adenophora similar to those produced by the commercial herbicide bromoxynil. Phytotoxicity bioassay tests using 24 plant species confirmed that citrinin has a broad activity spectrum and therefore has potential as a bioherbicide. Based on chlorophyll fluorescence studies, citrinin mainly blocks PSII electron flow beyond plastoquinone QA at the acceptor side, resulting in the inactivation of PSII reaction centers. Furthermore, molecular modeling of citrinin docking to the A. adenophora D1 protein suggests that it binds to the plastoquinone QB site by a hydrogen bond between the O1 hydroxy oxygen atom of citrinin and the histidine 215 of the D1 protein, the same way as classical phenolic PSII herbicides do. Finally, 32 new citrinin derivatives were designed and sorted according to free energies on the basis of the molecular model of an interaction between the citrinin molecule and the D1 protein. Five of the modeled compounds had much higher ligand binding affinity within the D1 protein compared with lead compound citrinin. CONCLUSION: Citrinin is a novel natural PSII inhibitor that has the potential to be developed into a bioherbicide or utilized as a lead compound for discovery of new derivatives with high herbicidal potency. © 2023 Society of Chemical Industry.

Effects of Mycotoxin Fumagillin, Mevastatin, Radicicol, and Wortmannin on Photosynthesis of Chlamydomonas reinhardtii.

Mycotoxins are one of the most important sources for the discovery of new pesticides and drugs because of their chemical structural diversity and fascinating bioactivity as well as unique novel targets. Here, the effects of four mycotoxins, fumagillin, mevastatin, radicicol, and wortmannin, on photosynthesis were investigated to identify their precise sites of action on the photosynthetic apparatus of Chlamydomonas reinhardtii. Our results showed that these four mycotoxins have multiple targets, acting mainly on photosystem II (PSII). Their mode of action is similar to that of diuron, inhibiting electron flow beyond the primary quinone electron acceptor (QA) by binding to the secondary quinone electron acceptor (QB) site of the D1 protein, thereby affecting photosynthesis. The results of PSII oxygen evolution rate and chlorophyll (Chl) a fluorescence imaging suggested that fumagillin strongly inhibited overall PSII activity; the other three toxins also exhibited a negative influence at the high concentration. Chl a fluorescence kinetics and the JIP test showed that the inhibition of electron transport beyond QA was the most significant feature of the four mycotoxins. Fumagillin decreased the rate of O2 evolution by interrupting electron transfer on the PSII acceptor side, and had multiple negative effects on the primary photochemical reaction and PSII antenna size. Mevastatin caused a decrease in photosynthetic activity, mainly due to the inhibition of electron transport. Both radicicol and wortmannin decreased photosynthetic efficiency, mainly by inhibiting the electron transport efficiency of the PSII acceptor side and the activity of the PSII reaction centers. In addition, radicicol reduced the primary photochemical reaction efficiency and antenna size. The simulated molecular model of the four mycotoxins' binding to C. reinhardtii D1 protein indicated that the residue D1-Phe265 is their common site at the QB site. This is a novel target site different from those of commercial PSII herbicides. Thus, the interesting effects of the four mycotoxins on PSII suggested that they provide new ideas for the design of novel and efficient herbicide molecules.

Structure-based ligand design and discovery of novel tenuazonic acid derivatives with high herbicidal activity.

INTRODUCTION: Computer-aided design has become an important tool to develop novel pesticides based on natural lead compounds. Tenuazonic acid (TeA), a typical representative of the natural tetramic acid family, was patented as a potential bioherbicide. However, its herbicidal efficacy is still not up to the ideal standard of commercial products. OBJECTIVES: We aim to find new TeA's derivatives with improved potency. METHODS: Molecular docking was used to build ligand-acceptor interaction models, design and screen new derivatives. Phytotoxicity, oxygen evolution rate, chlorophyll fluorescence and herbicidal efficacy were determined to estimate biological activity of compounds. RESULTS: With the aid of a constructed molecular model of natural lead molecule TeA binding to the QB site in Arabidopsis D1 protein, a series of derivatives differing in the alkyl side chain were designed and ranked according to free energies. All compounds are stabilized by hydrogen bonding interactions between their carbonyl oxygen O2 and D1-Gly256 residue; moreover, hydrogen bond distance is the most important factor for maintaining high binding affinity. Among 54 newly designed derivatives, D6, D13 and D27 with better affinities than TeA were screened out and synthesized to evaluate their photosynthetic inhibitory activity and herbicidal efficacy. Analysis of structure-activity relationship indicated that D6 and D13 with sec-pentyl and sec-hexyl side chains, respectively, were about twice more inhibitory of PSII activity and effective as herbicide than TeA with a sec-butyl side chain. CONCLUSION: D6 and D13 are promising compounds to develop TeA-derived novel PSII herbicides with superior performance.

Effect of Mycotoxin Cytochalasin A on Photosystem II in Ageratina adenophora.

Biological herbicides have received much attention due to their abundant resources, low development cost, unique targets and environmental friendliness. This study reveals some interesting effects of mycotoxin cytochalasin A (CA) on photosystem II (PSII). Our results suggested that CA causes leaf lesions on Ageratina adenophora due to its multiple effects on PSII. At a half-inhibitory concentration of 58.5 µΜ (I50, 58.5 µΜ), the rate of O2 evolution of PSII was significantly inhibited by CA. This indicates that CA possesses excellent phytotoxicity and exhibits potential herbicidal activity. Based on the increase in the J-step of the chlorophyll fluorescence rise OJIP curve and the analysis of some JIP-test parameters, similar to the classical herbicide diuron, CA interrupted PSII electron transfer beyond QA at the acceptor side, leading to damage to the PSII antenna structure and inactivation of reaction centers. Molecular docking model of CA and D1 protein of A. adenophora further suggests that CA directly targets the QB site of D1 protein. The potential hydrogen bonds are formed between CA and residues D1-His215, D1-Ala263 and D1-Ser264, respectively. The binding of CA to residue D1-Ala263 is novel. Thus, CA is a new natural PSII inhibitor. These results clarify the mode of action of CA in photosynthesis, providing valuable information and potential implications for the design of novel bioherbicides.

Overexpression of gamma-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: physiological and chlorophyll a fluorescence measurements.

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the gamma-tocopherol methyl transferase (gamma-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of alpha-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the gamma-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial "O" level to the "P" (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that alpha-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.

Action Mode of the Mycotoxin Patulin as a Novel Natural Photosystem II Inhibitor.

A biocontrol method plays an important role in weed management. In this study, we aimed to clarify the phytotoxicity of the mycotoxin patulin (PAT) and reveal its mode of action as a new natural photosystem II (PSII) inhibitor. Phytotoxicity test showed that PAT has herbicidal activity and causes significant leaf lesions on Ageratina adenophora. Under a half-inhibition concentration I50 (2.24 µM), the observed significant decrease in oxygen evolution rate and the increase in the J-step of the chlorophyll fluorescence rise OJIP curve indicated that PAT strongly reduces photosynthetic efficiency by blocking electron transport from the primary to secondary plastoquinone acceptors (QA to QB) of PSII. Molecular modeling of PAT docking to the A. adenophora D1 protein suggested that PAT bounds to the QB site by forming hydrogen bonds to histidine 252 in the D1 protein. It is proposed that PAT is a new natural PSII inhibitor and has the potential to be developed into a bioherbicide or used as a template scaffold for discovering novel derivatives with more potent herbicidal activity.

Tenuazonic Acid-Triggered Cell Death Is the Essential Prerequisite for Alternaria alternata (Fr.) Keissler to Infect Successfully Host Ageratina adenophora.

The necrotrophic fungus Alternaria alternata contains different pathotypes that produce different mycotoxins. The pathotype Ageratina adenophora secretes the non-host-selective toxin tenuazonic acid (TeA), which can cause necrosis in many plants. Although TeA is thought to be a central virulence factor of the A. adenophora pathotype, the precise role of TeA in different stages of host infection by pathogens remains unclear. Here, an A. alternata wild-type and the toxin-deficient mutant ΔHP001 with a 75% reduction in TeA production were used. It was observed that wild-type pathogens could induce the reactive oxygen species (ROS) bursts in host leaves and killed photosynthetic cells before invading hyphae. The ROS interceptor catalase remarkably inhibited hyphal penetration and invasive hyphal growth and expansion in infected leaves and suppressed necrotic leaf lesion. This suggests that the production of ROS is critical for pathogen invasion and proliferation and disease symptom formation during infection. It was found that the mutant pathogens did not cause the formation of ROS and cell death in host leaves, showing an almost complete loss of disease susceptibility. In addition, the lack of TeA resulted in a significant reduction in the ability of the pathogen to penetrate invasive hyphal growth and spread. The addition of exogenous TeA, AAL-toxin, and bentazone to the mutant ΔHP001 pathogens during inoculation resulted in a significant restoration of pathogenicity by increasing the level of cell death, frequency of hyphal penetration, and extent of invasive hyphal spread. Our results suggest that cell death triggered by TeA is the essential requirement for successful colonization and disease development in host leaves during infection with A. adenophora pathogens.

Comparative effect of tenuazonic acid, diuron, bentazone, dibromothymoquinone and methyl viologen on the kinetics of Chl a fluorescence rise OJIP and the MR820 signal.

In this study, the comparative effect of TeA, DCMU, bentazone, DBMIB and MV on prompt fluorescence and the MR820 signal was simultaneously analyzed to provide an insight into how to elucidate their precise influence on Ageratina adenophora photosystems. The herbicides that interrupt electron transport beyond QA, such as TeA, DCMU and bentazone, mainly increased the J-step level of fluorescence rise kinetics as a result of accumulation of QA-, but showed differences in detail. The IP phase disappeared in the presence of DCMU and bentazone with a significant increase in FO value. TeA treatment retained the IP phase with lowering FM. As an inhibitor of plastoquinone re-oxidation, DBMIB increased the I-step (IP phase almost unnoticable) without changing FO and FM values. MV blocking PSI electron transfer through intercepting electrons from the FeS clusters suppressed the IP phase by decreasing the P level. Considering the WIP kinetics, TeA and DBMIB also affected PSI activity. After DCMU and MV treatment, the major change in the MR820 kinetics was the loss of the slow phase due to the complete prevention of electron movement from PSII to re-reduce PC+ and P700+. TeA, bentazone and DBMIB clearly suppressed the MR820 slow phase and decreased the re-reduction rate of PC+ and P700+ (Vred), significantly. However, there were still parts of electrons being donated to PC+ and P700+, showing a smaller slow phase and PC+ and P700+ re-reduction rate. Additionally, TeA and DBMIB also somewhat declined the fast phase and PC and P700 oxidation rate (Vox).

Selection of tree species for forests under climate change: is PSI functioning a better predictor for net photosynthesis and growth than PSII?

A chlorophyll fluorescence (ChlF) assessment was carried out on oak seedlings (Quercus ilex L., Quercus pubescens Willd., Quercus frainetto Ten.) of Italian and Greek provenance, during the years 2017 and 2018, in a common garden in central Italy planted in 2017. This trial aimed to test the relative performances of the oak species in the perspective of assisted migration as part of the actions for the adaptation of forests to climate change. The assessment of the photosynthetic performance of the tree species included the analysis of the prompt chlorophyll fluorescence (PF) transient and the modulated reflection (MR) at 820 nm, leaf chlorophyll content, leaf gas exchange (net photosynthesis, stomatal conductance), plant growth (i.e., height) and mortality rate after 2 years from the beginning of the experiment. The assessment of the performance of the three oak species was carried out 'in vivo'. Plants were generated from seeds and exposed to several environmental factors, including changing seasonal temperature, water availability, and soil biological and physical functionality. The results of PF indicate a stable functionality of the photosynthetic system PSII (expressed as FV/FM) across species and provenances and a decline in photochemistry functionality at the I-P phase (ΔVIP) in Q. frainetto, thus indicating a decline of the content of PSI in this species. This result was confirmed by the findings of MR analysis, with the speed of reduction and subsequent oxidation of PSI (VRED and VOX) strongly correlated to the amplitude of ΔVIP. The photosynthetic rates (net photosynthesis, PN) and growth were correlated with the parameters associated with PSI content and function, rather than those related to PSII. The low performance of Q. frainetto in the common garden seems to be related to early foliar senescence with the depletion of nitrogen, due to suboptimal climatic and edaphic conditions. Chlorophyll fluorescence allowed discrimination of populations of oak species and individuation of the less (or/and best) suitable species for future forest ecology and management purposes.

Novel Action Targets of Natural Product Gliotoxin in Photosynthetic Apparatus.

Gliotoxin (GT) is a fungal secondary metabolite that has attracted great interest due to its high biological activity since it was discovered by the 1930s. It exhibits a unique structure that contains a N-C = O group as the characteristics of the classical PSII inhibitor. However, GT's phytotoxicity, herbicidal activity and primary action targets in plants remain hidden. Here, it is found that GT can cause brown or white leaf spot of various monocotyledonous and dicotyledonous plants, being regarded as a potential herbicidal agent. The multiple sites of GT action are located in two photosystems. GT decreases the rate of oxygen evolution of PSII with an I 50 value of 60 µM. Chlorophyll fluorescence data from Chlamydomonas reinhardtii cells and spinach thylakoids implicate that GT affects both PSII electron transport at the acceptor side and the reduction rate of PSI end electron acceptors' pool. The major direct action target of GT is the plastoquinone QB-site of the D1 protein in PSII, where GT inserts in the QB binding niche by replacing native plastoquinone (PQ) and then interrupts electron flow beyond plastoquinone QA. This leads to severe inactivation of PSII RCs and a significant decrease of PSII overall photosynthetic activity. Based on the simulated modeling of GT docking to the D1 protein of spinach, it is proposed that GT binds to the-QB-site through two hydrogen bonds between GT and D1-Ser264 and D1-His252. A hydrogen bond is formed between the aromatic hydroxyl oxygen of GT and the residue Ser264 in the D1 protein. The 4-carbonyl group of GT provides another hydrogen bond to the residue D1-His252. So, it is concluded that GT is a novel natural PSII inhibitor. In the future, GT may have the potential for development into a bioherbicide or being utilized as a lead compound to design more new derivatives.

Comparative phytotoxicity of usnic acid, salicylic acid, cinnamic acid and benzoic acid on photosynthetic apparatus of Chlamydomonas reinhardtii.

The effects of four phytotoxins usnic acid (UA), salicylic acid (SA), cinnamic acid (CA) and benzoic acid (BA) on photosynthesis of Chlamydomonas reinhardtii were studied in vivo to identify and localise their initial action sites on two photosystems. Our experimental evidence shows that the four phytotoxins have multiple targets in chloroplasts, which mainly lie in photosystem II (PSII), not photosystem I (PSI). They share an original action site by blocking electron transport beyond QA (primary plastoquinone acceptor) at PSII acceptor side since a fast increase of the J-step level is the greatest change in chlorophyll a fluorescence induction kinetics OJIP in C. reinhardtii cells treated with the phytotoxins. UA decreases photosynthetic activity by reducing O2 evolution rate, interrupting PSII electron transport at both the donor and acceptor sides, inactivating the PSII reaction centers (RCs), reducing the content of chlorophylls and carotenoids, destroying the conformation of antenna pigment assemblies, and casuing the degradation of D1/D2 proteins. SA damage to photosynthetic machinery is mainly attributed to inhibition of PSII electron transport beyond QA at the acceptor side, inactivation of the PSII RCs, reduction of chlorophyll content, digestion of thylakoid ploypeptides and destabilization of thylakoid membranes. Both CA and BA affect the photosynthetic process by decreasing PSII electron transport efficiency at the acceptor side and the amount of active PSII RCs. Besides, the initial cause of BA-inhibiting photosynthesis is also assocaited with the O2 evolution rate and the disconnection of some antenna molecules from PSII RCs.

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A.

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions.

In vivo assessment of effect of phytotoxin tenuazonic acid on PSII reaction centers.

Tenuazonic acid (TeA), a phytotoxin produced by the fungus Alternaria alternata isolated from diseased croftonweed (Ageratina adenophora), exhibits a strong inhibition in photosystem II (PSII) activity. In vivo chlorophyll fluorescence transients of the host plant croftonweed, show that the dominant effect of TeA is not on the primary photochemical reaction but on the biochemical reaction after QA. The most important action site of TeA is the QB site on the PSII electron-acceptor side, blocking electron transport beyond QA(-) by occupying the QB site in the D1 protein. However, TeA does not affect the antenna pigments, the energy transfer from antenna pigment molecules to reaction centers (RCs), and the oxygen-evolving complex (OEC) at the donor side of PSII. TeA severely inactivated PSII RCs. The fraction of non-QA reducing centers and non-QB reducing centers show a time- and concentration-dependent linear increase. Conversely, the amount of active QA or QB reducing centers declined sharply in a linear way. The fraction of non-QB reducing centers calculated from data of fluorescence transients is close to the number of PSII RCs with their QB site filled by TeA. An increase of the step-J level (VJ) in the OJIP fluorescence transients attributed to QA(-) accumulation due to TeA bound to the QB site is a typical characteristic response of the plants leaf with respect to TeA penetration.

Effect of vulculic acid produced by Nimbya alternantherae on the photosynthetic apparatus of Alternanthera philoxeroides.

The effect of the toxin vulculic acid produced by Nimbya alternantherae, on the photosynthetic apparatus of Alternanthera philoxeroides, was investigated via the photochemical activity and SDS-PAGE of protein on thylakoid membranes, fast chlorophyll a fluorescence transient measurements and the JIP-test. The electron transport rate of photosystem II (PSII), non-cyclic photophosphorylation activity, as well as the activity of chloroplast ATPase and Rubisco reduced significantly after vulculic acid treatment. Vulculic acid affected the O-J-I-P fluorescence induction kinetics, showing an increase of the parameters FV/FO, VK and VJ and a decrease of FO, FM, PIABS, φPo, ψEo, φEo, φRo, δRo and PItotal. In addition, it significantly decreased the amounts of major photosystem I (PSI) and PSII proteins. It is concluded that vulculic acid is a photosynthetic inhibitor with multiple action sites. The main targets are the light harvesting complex (LHC) and the oxygen evolving complex (OEC) on the PSII donor side. Vulculic acid blocks electron transport beyond QA and on the PSI acceptor side by digesting major PSI and PSII proteins.

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.

Preservation of photosynthetic electron transport from senescence-induced inactivation in primary leaves after decapitation and defoliation of bean plants.

The comparative effects of decapitation and defoliation on the senescence-induced inactivation of photosynthetic activity in primary leaves of bean plants were investigated. Decapitation was performed during different phases of bean plant ontogenesis, immediately after the appearance of the 1st, 2nd, 3rd and 4th composite leaf. In addition, we examined a variant with primary leaves and stem with an apical bud, but without composite leaves, i.e. defoliated plants. Analyses of chlorophyll fluorescence, millisecond delayed fluorescence and absorption at 830nm in primary leaves were undertaken to investigate the alterations in photosystems II and I electron transport during the decapitation-induced delayed senescence in the non-detached leaves. Analysis of the OKJIP transients using the JIP-test (see [Strasser R, Srivastava A, Tsimilli-Michael M. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G, Govindjee, editors. Chlorophyll a fluorescence: a signature of photosynthesis. The Netherlands: Kluwer Academic Publishers, 2004; pp. 321-362]) showed an increase in several biophysical parameters of photosystem II in decapitated plants, specifically, the density of active reaction centers on a chlorophyll basis, the yields of trapping and electron transport, and the performance index. We also observed a decrease in the absorbed light energy per reaction center. Such a decrease in light absorption could be a result of the photosystem II down regulation that appeared as an increase in Q(B)-non-reducing photosystem II centers. The effect was identical when all leaves except the primary leaves were removed. The variant with a preserved apical bud, the defoliated plant, showed values similar to those of decapitated plants with primary leaves only. The changes in the induction curves of the delayed fluorescence also indicated an acceleration of electron transport beyond photosystem II in the decapitated and in defoliated plants. In these plants, the photosystem I-driven electron transport was accelerated, and the size of the plastoquinone pool was enhanced. It was established that decapitation can retard the senescence of primary leaves, can expand leaf life span and can cause activation of both photosystems I and II electron transport. The decapitation procedure shows similarities to the process of defoliation. The overcompensation effect that is developed after defoliation could initially be manifested as an acceleration of the linear photosynthetic electron flow in the rest of the leaves.