Reactive Oxygen Species In Chloroplasts Research Articles

Salicylic acid- and ethylene-dependent effects of the ER stress-inducer tunicamycin on the photosynthetic light reactions in tomato plants

Plant hormones such as ethylene (ET) and salicylic acid (SA) have an elementary role in the regulation of ER stress and unfolded protein response (UPR) in plants via modulating defence responses or inducing oxidative stress. Chloroplasts can be sources and targets of reactive oxygen species (ROS) that affect photosynthetic efficiency, which has not been investigated under tunicamycin (Tm)-induced ER stress. In this study, the direct and indirect effects of Tm on chloroplastic ROS production were first investigated in leaves of wild-type tomato (Solanum lycopersicum L.) plants. Secondly changes in activities of photosystem II and I were analysed under Tm exposure and after application of the chemical chaperone 4-phenylbutyrate (PBA) in different genotypes, focusing on the regulatory role of SA and ET Tm treatments significantly but indirectly induced ROS production in tomato leaves and in parallel it decreased the effective quantum yield of PSII [Y(II)] and PSI [Y(I)], as well as the photochemical quenching coefficient (qP) and the quantum yield of non-photochemical energy dissipation in PSI due to acceptor-side limitation [Y(NA)]. At the same time, Tm increased non-photochemical quenching (NPQ) and cyclic electron flow (CEF) in tomato leaves after 24 h. However, the photosynthetic activity of the SA hydroxylase-overexpressing NahG tomato plants was more severely affected by Tm as compared to wild-type and ET-insensitive Never ripe (Nr) plants. These results suggest the protective role of SA in the regulation of photosynthetic activity contributing to UPR and the survival of plants under ER stress. Interestingly, the activation of photoprotective mechanisms by NPQ was independent of SA but dependent on active ET signalling under ER stress, whereas CEF was reduced by ET due to its higher ratio in Nr plants.

Methyl viologen-induced changes in the Arabidopsis proteome implicate PATELLIN 4 in oxidative stress responses.

The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

Inhibition of invasive plant Mikania micrantha rapid growth by host-specific rust (Puccinia spegazzinii).

Mikania micrantha Kunth is a fast-growing global invasive weed species that causes severe damage to natural ecosystems and very large economic losses of forest and crop production. Although Puccinia spegazzinii can effectively inhibit the growth of M. micrantha and is used as a biological control strain in many countries, the mechanism of inhibiting the growth in Mikania micrantha is not clear. Here, we used a combination of phenotypic, enzyme activity, transcriptomic and metabolomic approaches to study the response of M. micrantha after infection by P. spegazzinii. In the early stages of rust infection, JA, JA-Ile and SA levels in infected leaves were significantly lower than that in uninfected leaves. In teliospore initial and developed stages of P. spegazzinii, JA and JA-Ile levels substantially increased by more than 6 times, which resulted in a significant decrease in accumulation of defense hormone SA in infected leaves of M. micrantha. The contents of plant growth-promoting hormones were significantly reduced in the infected plants as a result of substantial downregulation of expression of key genes related to hormone biosynthesis. Furthermore, rust infection led to high levels of ROS in chloroplasts and the destruction of chlorophyll structure, which also led to decreased photosynthetic gene expression, net photosynthetic rate, activity of Rubisco and levels of important organic acids in the Calvin cycle. We hypothesised that after P. spegazzinii infection, JA or JA-Ile accumulation not only inhibited SA levels to promote rust infection and development, but also impeded the rapid growth of M. micrantha by affecting plant growth hormones, carbon, and nitrogen metabolic pathways.

Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants.

Pepper mild mottle virus (PMMoV) is a devastating viral pathogen of pepper (Capsicum annuum) but it is unclear whether and how peppers protect against PMMoV infection. The expression of the chloroplast outer membrane protein 24 (OMP24) of C. annuum was upregulated under PMMoV infection and it interacted with PMMoV coat protein (CP). Silencing of OMP24 in either C. annuum or Nicotiana benthamiana facilitated PMMoV infection, whereas overexpression of N. benthamiana OMP24 in transgenic plants inhibited PMMoV infection. Both C. annuum OMP24 (CaOMP24) and N. benthamiana OMP24 (NbOMP24) localized to the chloroplast and have a moderately hydrophobic transmembrane domain that is necessary for their localization. Overexpression of CaOMP24 induced stromules, perinuclear chloroplast clustering, and accumulation of reactive oxygen species (ROS), the typical defense responses of chloroplasts transferring the retrograde signaling to the nucleus to regulate resistance genes. The expression of PR1 and PR2 was also upregulated significantly in plants overexpressing OMP24. Self-interaction of OMP24 was demonstrated and was required for OMP24-mediated plant defense. Interaction with PMMoV CP interfered with the self-interaction of OMP24 and impaired OMP24-induced stromules, perinuclear chloroplast clustering and ROS accumulation. The results demonstrate the defense function of OMP24 in pepper during viral infection and suggest a possible mechanism by which PMMoV CP modulates the plant defense to facilitate viral infection.

Cytoplasmic regulation of chloroplast ROS accumulation during effector-triggered immunity.

Accumulating evidence suggests that chloroplasts are an important battleground during various microbe-host interactions. Plants have evolved layered strategies to reprogram chloroplasts to promote de novo biosynthesis of defense-related phytohormones and the accumulation of reactive oxygen species (ROS). In this minireview, we will discuss how the host controls chloroplast ROS accumulation during effector-triggered immunity (ETI) at the level of selective mRNA decay, translational regulation, and autophagy-dependent formation of Rubisco-containing bodies (RCBs). We hypothesize that regulation at the level of cytoplasmic mRNA decay impairs the repair cycle of photosystem II (PSII) and thus facilitates ROS generation at PSII. Meanwhile, removing Rubisco from chloroplasts potentially reduces both O2 and NADPH consumption. As a consequence, an over-reduced stroma would further exacerbate PSII excitation pressure and enhance ROS production at photosystem I.

Chloroplast redox state changes mark cell-to-cell signaling in the hypersensitive response.

Hypersensitive response (HR)-conferred resistance is associated with induction of programmed cell death and pathogen spread restriction in its proximity. The exact role of chloroplastic reactive oxygen species and its link with salicylic acid (SA) signaling in HR remain unexplained. To unravel this, we performed a detailed spatiotemporal analysis of chloroplast redox response in palisade mesophyll and upper epidermis to potato virus Y (PVY) infection in a resistant potato genotype and its transgenic counterpart with impaired SA accumulation and compromised resistance. Besides the cells close to the cell death zone, we detected individual cells with oxidized chloroplasts further from the cell death zone. These are rare in SA-deficient plants, suggesting their role in signaling for resistance. We confirmed that chloroplast redox changes play important roles in signaling for resistance, as blocking chloroplast redox changes affected spatial responses at the transcriptional level. Through spatiotemporal study of stromule induction after PVY infection, we show that stromules are induced by cell death and also as a response to PVY multiplication at the front of infection. Overall induction of stromules is attenuated in SA-deficient plants.

Chloroplasts play a central role in facilitating MAMP-triggered immunity, pathogen suppression of immunity and crosstalk with abiotic stress.

Microbe‐associated molecular pattern (MAMP)‐triggered immunity (MTI) research has traditionally centred around signal transduction pathways originating from activated membrane‐localized pattern recognition receptors (PRRs), culminating in nuclear transcription and posttranslational modifications. More recently, chloroplasts have emerged as key immune signalling hubs, playing a central role in integrating environmental signals. Notably, MAMP recognition induces chloroplastic reactive oxygen species (cROS) that is suppressed by pathogen effectors, which also modify the balance of chloroplast‐synthesized precursors of the defence hormones, jasmonic acid, salicylic acid (SA) and abscisic acid. This study focuses on how well‐characterized PRRs and coreceptors modulate chloroplast physiology, examining whether diverse signalling pathways converge to similarly modulate chloroplast function. Pretreatment of receptor mutant plants with MAMP and D(Damage)AMP peptides usually protect against effector modulation of chlorophyll fluorescence and prevent Pseudomonas syringae effector‐mediated quenching of cROS and suppression of maximum dark‐adapted quantum efficiency (the ratio of variable/maximum fluorescence [F v/F m]). The MTI coreceptor double mutant, bak1‐5/bkk1‐1, exhibits a remarkable decrease in F v/F m compared to control plants during infection, underlining the importance of MTI‐mediated signalling in chloroplast immunity. Further probing the role of the chloroplast in immunity, we unexpectedly found that even moderate changes in light intensity can uncouple plant immune signalling.

Simultaneous Ozone and High Light Treatments Reveal an Important Role for the Chloroplast in Co-ordination of Defense Signaling.

Plants live in a world of changing environments, where they are continuously challenged by alternating biotic and abiotic stresses. To transfer information from the environment to appropriate protective responses, plants use many different signaling molecules and pathways. Reactive oxygen species (ROS) are critical signaling molecules in the regulation of plant stress responses, both inside and between cells. In natural environments, plants can experience multiple stresses simultaneously. Laboratory studies on stress interaction and crosstalk at regulation of gene expression, imply that plant responses to multiple stresses are distinctly different from single treatments. We analyzed the expression of selected marker genes and reassessed publicly available datasets to find signaling pathways regulated by ozone, which produces apoplastic ROS, and high light treatment, which produces chloroplastic ROS. Genes related to cell death regulation were differentially regulated by ozone versus high light. In a combined ozone + high light treatment, the light treatment enhanced ozone-induced cell death in leaves. The distinct responses from ozone versus high light treatments show that plants can activate stress signaling pathways in a highly precise manner.

Transcriptomic Data Meta-Analysis Sheds Light on High Light Response in Arabidopsis thaliana L.

The availability and intensity of sunlight are among the major factors of growth, development and metabolism in plants. However, excessive illumination disrupts the electronic balance of photosystems and leads to the accumulation of reactive oxygen species in chloroplasts, further mediating several regulatory mechanisms at the subcellular, genetic, and molecular levels. We carried out a comprehensive bioinformatic analysis that aimed to identify genetic systems and candidate transcription factors involved in the response to high light stress in Arabidopsis thaliana L. using resources GEO NCBI, string-db, ShinyGO, STREME, and Tomtom, as well as programs metaRE, CisCross, and Cytoscape. Through the meta-analysis of five transcriptomic experiments, we selected a set of 1151 differentially expressed genes, including 453 genes that compose the gene network. Ten significantly enriched regulatory motifs for TFs families ZF-HD, HB, C2H2, NAC, BZR, and ARID were found in the promoter regions of differentially expressed genes. In addition, we predicted families of transcription factors associated with the duration of exposure (RAV, HSF), intensity of high light treatment (MYB, REM), and the direction of gene expression change (HSF, S1Fa-like). We predicted genetic components systems involved in a high light response and their expression changes, potential transcriptional regulators, and associated processes.

The chloroplast redox-responsive transcriptome of solanaceous plants reveals significant nuclear gene regulatory motifs associated to stress acclimation.

Transcriptomes of solanaceous plants expressing a plastid-targeted antioxidant protein were analysed to identify chloroplast redox networks modulating the expression of nuclear genes associated with stress acclimation. Plastid functions depend on the coordinated expression of nuclear genes, many of them associated to developmental and stress response pathways. Plastid-generated signals mediate this coordination via retrograde signaling, which includes sensing of chloroplast redox state and levels of reactive oxygen species (ROS), although it remains a poorly understood process. Chloroplast redox poise and ROS build-up can be modified by recombinant expression of a plastid-targeted antioxidant protein, i.e., cyanobacterial flavodoxin, with the resulting plants displaying increased tolerance to multiple environmental challenges. Here we analysed the transcriptomes of these flavodoxin-expressing plants to study the coordinated transcriptional responses of the nucleus to the chloroplast redox status and ROS levels during normal growth and stress responses (drought or biotic stress) in tobacco and potato, members of the economically important Solanaceae family. We compared their transcriptomes against those from stressed and mutant plants accumulating ROS in different subcellular compartments and found distinct ROS-related imprints modulated by flavodoxin expression and/or stress. By introducing our datasets in a large-scale interaction network, we identified transcriptional factors related to ROS and stress responses potentially involved in flavodoxin-associated signaling. Finally, we discovered identical cis elements in the promoters of many genes that respond to flavodoxin in the same direction as in wild-type plants under stress, suggesting a priming effect of flavodoxin before stress manifestation. The results provide a genome-wide picture illustrating the relevance of chloroplast redox status on biotic and abiotic stress responses and suggest new cis and trans targets to generate stress-tolerant solanaceous crops.

Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis

The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, β-CA1 and β-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δβ-ca1 and Δβ-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δβ-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δβ-ca1ca5 plants normalized at 9,000 ppm CO2 Leaves of Δβ-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δβ-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δβ-ca1ca5 seedling germination and development is negatively affected at ambient CO2 Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented the Δβ-ca1ca5 mutation but inactivated (ΔZn-βCA1) and cytoplasm-localized (Δ62-βCA1) forms of β-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-βCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δβ-ca1 and Δβ-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.

EGY3 mediates chloroplastic ROS homeostasis and promotes retrograde signaling in response to salt stress in Arabidopsis.

The chloroplast is the main organelle for stress-induced production of reactive oxygen species (ROS). However, how chloroplastic ROS homeostasis is maintained under salt stress is largely unknown. We show that EGY3, a gene encoding a chloroplast-localized protein, is induced by salt and oxidative stresses. The loss of EGY3 function causes stress hypersensitivity while EGY3 overexpression increases the tolerance to both salt and chloroplastic oxidative stresses. EGY3 interacts with chloroplastic Cu/Zn-SOD2 (CSD2) and promotes CSD2 stability under stress conditions. In egy3-1 mutant plants, the stress-induced CSD2 degradation limits H2O2 production in chloroplasts and impairs H2O2-mediated retrograde signaling, as indicated by the decreased expression of retrograde-signal-responsive genes required for stress tolerance. Both exogenous application of H2O2 (or APX inhibitor) and CSD2 overexpression can rescue the salt-stress hypersensitivity of egy3-1 mutants. Our findings reveal that EGY3 enhances the tolerance to salt stress by promoting the CSD2 stability and H2O2-mediated chloroplastic retrograde signaling.

Plastid-mediated singlet oxygen in regulated cell death.

Reactive oxygen species (ROS) generation within a cell is a natural process of specific subcellular components involved in redox reactions. Within a plant cell, chloroplasts are one of the major sources of ROS generation. Plastid-generated ROS molecules include singlet oxygen (1 O2 ), superoxide radical (O2 - ), hydroxyl radical (OH• ) and hydrogen peroxide (H2 O2 ), which are produced mainly during photochemical reactions of photosynthesis and chlorophyll biosynthetic process. Under normal growth and developmental, generated ROS molecules act as a secondary messenger controlling several metabolic reactions; however, perturbed environmental conditions lead to multi-fold amplification of cellular ROS that eventually kill the target cell. To maintain homeostasis between production and scavenging of ROS, the cell has instituted several enzymatic and non-enzymatic antioxidant machineries to maintain ROS at a physiological level. Among chloroplastic ROS molecules, excess generation of singlet oxygen (1 O2 ) is highly deleterious to the cell metabolic functions and survival. Interestingly, within cellular antioxidant machinery, enzymes involved in detoxification of 1 O2 are lacking. Recent studies suggest that under optimal concentrations, 1 O2 acts as a signalling molecule and drives the cell to either the acclimation pathway or regulated cell death (RCD). Stress-induced RCD is a survival mechanism for the whole plant, while the involvement of chloroplasts and chloroplast-localized molecules that execute RCD are not well understood. In this review, we advocate for participation of chloroplasts-generated 1 O2 in signalling and RCD in plants.

Age-dependent increase in α-tocopherol and phytosterols in maize leaves exposed to elevated ozone pollution.

Tropospheric ozone is a major air pollutant that significantly damages crop production. Crop metabolic responses to rising chronic ozone stress have not been well studied in the field, especially in C4 crops. In this study, we investigated the metabolomic profile of leaves from two diverse maize (Zea mays) inbred lines and the hybrid cross during exposure to season‐long elevated ozone (~100 nl L−1) in the field using free air concentration enrichment (FACE) to identify key biochemical responses of maize to elevated ozone. Senescence, measured by loss of chlorophyll content, was accelerated in the hybrid line, B73 × Mo17, but not in either inbred line (B73 or Mo17). Untargeted metabolomic profiling further revealed that inbred and hybrid lines of maize differed in metabolic responses to ozone. A significant difference in the metabolite profile of hybrid leaves exposed to elevated ozone occurred as leaves aged, but no age‐dependent difference in leaf metabolite profiles between ozone conditions was measured in the inbred lines. Phytosterols and α‐tocopherol levels increased in B73 × Mo17 leaves as they aged, and to a significantly greater degree in elevated ozone stress. These metabolites are involved in membrane stabilization and chloroplast reactive oxygen species (ROS) quenching. The hybrid line also showed significant yield loss at elevated ozone, which the inbred lines did not. This suggests that the hybrid maize line was more sensitive to ozone exposure than the inbred lines, and up‐regulated metabolic pathways to stabilize membranes and quench ROS in response to chronic ozone stress.

Effects of excess ammoniacal nitrogen (NH4+-N) on pigments, photosynthetic rates, chloroplast ultrastructure, proteomics, formation of reactive oxygen species and enzymatic activity in submerged plant Hydrilla verticillata (L.f.) Royle

Although excess ammoniacal-nitrogen (NH4+-N) results in the disturbance of various important biochemical and physiological processes, a detailed study on the effects of NH4+-N stress on the photosynthesis and global changes in protein levels in submerged macrophytes is still lacking. Here, the changes of excess NH4+-N on physiological parameters in Hydrilla verticillata (L.f.) Royle, a submerged macrophyte were investigated, including the contents of photosynthetic pigments, soluble sugars, net photosynthesis and respiration, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, chloroplast ultrastructure, chloroplast reactive oxygen species (ROS) accumulation and protein levels. Our results showed that the net photosynthetic rate and pigment content reached maximum values when the plants were treated with 1 and 2 mg L-1 NH4+-N, respectively, and decreased at NH4+-N concentrations at 5, 10, 15 and 20 mg L-1. This decrease might be caused by ROS accumulation. Compared that in 0.02 mg L-1 NH4+-N as a control, ROS generation in chloroplasts significantly increased in the presence of more than 2 mg L-1 NH4+-N. Consistently, the damages caused by over-accumulated ROS were observed in chloroplast ultrastructure, showing a loose thylakoid membranes and swollen grana/stroma lamellae. Furthermore, through proteomic analysis, we identified 91 differentially expressed protein spots. Among them, six proteins involved in photosynthesis decreased in abundance in response to excess NH4+-N. Surprisingly, the abundance of all the identified proteins that were involved in nitrogen assimilation and amino acid metabolism tended to increase under excess NH4+-N compared with the control, suggestive of the imbalanced carbon and nitrogen (C-N) metabolisms. In support, activated GS and GOGAT cycle was observed, evidenced by higher activities of GS and GOGAT enzymes. To our knowledge, this work is the first description that excess NH4+-N results in chloroplast ultrastructural damages and the first proteomic evidence to support that excess NH4+-N can lead to a decline in photosynthesis and imbalance of C-N metabolism in submerged macrophytes.

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.

Disturbance of photosystem II-oxygen evolution complex induced the oxidative damage in Chlorella vulgaris under the stress of cetyltrimethylammonium chloride

Oxygen evolution complex (OEC) in photosystem II (PSII) is sensitive to environmental stressors. However, oxidative damage mechanism in PSII-OEC is still unclear. Here, we investigated photosynthetic performance of PSII, oxidative stress and antioxidant reaction induced by reactive oxygen species (ROS) in a unicellular green alga Chlorella vulgaris (C. vulgaris) under the stress of cetyltrimethylammonium chloride (CTAC). From the changes of chlorophyll fluorescence parameters and PSII activity, it was proved that the electron transport, which occurred initially at the electron donor side of OEC, was disturbed by CTAC. Moreover, a significant decrease of the oxygen evolution rate in OEC (40.95%) while an increase of ROS (50.50%) was obtained after the exposure to 0.6 mg/L CTAC compared to the control (without CTAC), confirming that more oxygen transferred to ROS under the stress. Furthermore, the increased ROS in chloroplast and the structural destruction in thylakoid membrane were observed, respectively. These results proved that oxidative damage mechanism in PSII-OEC is mainly through the reduction of oxygen evolution and the production of excessive ROS, thus leading to the destruction of OEC performance and chloroplast structure.

Mechanism of long-term toxicity of CuO NPs to microalgae

Little is known regarding the detailed mechanism of CuO NPs’ toxicity to microalgal primary metabolism pathway. Photosynthesis and respiration are the most important primary metabolism and the main sources of production of reactive oxygen species (ROS), but the effect of CuO NPs on both of them has not been systematically studied to date. Our research demonstrated that long-term treatment with CuO NPs significantly inhibited activities of photosynthesis and respiration in microalgae, and the photosynthesis was more sensitive to the toxicity of CuO NPs than respiration. CuO NPs could be absorbed by microalgae and be converted into Cu2O NPs concentrated in chloroplast. The internalized Cu, regardless of whether the exposure was Cu2+ or CuO NPs had the same capacity to damage chloroplast structure. The result also shows that the oxygen-evolving complex (OEC) in the photosynthetic electron transport chain was the most sensitive site to CuO NPs and Cu2+-treated microalgae had the same damage site as that of CuO NPs, which may be related to the Mn cluster that is dissociated by Cu ions released from CuO NPs. The damage of OEC inhibited photosynthetic electron transport to increase excess excited energy, which caused the accumulation of ROS in chloroplast. The accumulation of ROS damaged the structure of cell membrane and aggravated the PSII photoinhibition, further decreasing the efficiency of light energy utilization. In conclusion, the Cu ionic toxicity of photosynthetic apparatus by CuO NPs resulted in the carbon starvation and the accumulation of ROS to inhibit the growth of microalgae.

Expression of a Plastid-Targeted Flavodoxin Decreases Chloroplast Reactive Oxygen Species Accumulation and Delays Senescence in Aging Tobacco Leaves.

Leaf senescence is a concerted physiological process involving controlled degradation of cellular structures and reallocation of breakdown products to other plant organs. It is accompanied by increased production of reactive oxygen species (ROS) that are proposed to signal cell death, although both the origin and the precise role of ROS in the execution of this developmental program are still poorly understood. To investigate the contribution of chloroplast-associated ROS to natural leaf senescence, we used tobacco plants expressing a plastid-targeted flavodoxin, an electron shuttle flavoprotein present in prokaryotes and algae. When expressed in plants, flavodoxin specifically prevents ROS formation in chloroplasts during stress situations. Senescence symptoms were significantly mitigated in these transformants, with decreased accumulation of chloroplastic ROS and differential preservation of chlorophylls, carotenoids, protein contents, cell and chloroplast structures, membrane integrity and cell viability. Flavodoxin also improved maintenance of chlorophyll-protein complexes, photosynthetic electron flow, CO2 assimilation, central metabolic routes and levels of bioactive cytokinins and auxins in aging leaves. Delayed induction of senescence-associated genes indicates that the entire genetic program of senescence was affected by flavodoxin. The results suggest that ROS generated in chloroplasts are involved in the regulation of natural leaf senescence.

Effect of Temperature Stress on Antioxidant Defenses in Brassica oleracea.

Brassica oleracea crops are exposed to seasonal changes in temperature because of their biennial life cycle. Extreme temperatures (cold and heat) affect the photosynthetic activity and the yield of cabbage (B. oleracea capitata group) and kale (B. oleracea acephala group). We studied the relationship among antioxidant defenses, photosynthesis, and yield under extreme temperatures in both crops. Under these conditions, the plants increase the antioxidant defenses, responding to an increment in reactive oxygen species (ROS). The accumulation of ROS in chloroplasts decreases the chlorophyll content and provokes photoinhibition that leads to a low fixation of CO2 and loss of dry weight. Low temperatures especially increase the antioxidant defenses and decrease the chlorophyll content compared to the heat conditions. However, dry weight losses are higher when plants are grown under heat than under cold conditions, probably because of the inactivation of Rubisco and/or the associated enzymes. Both crops were more resilient to cold than to heat temperatures, the capitata group being more resistant.