Cross Talk between Hydrogen Peroxide and Nitric Oxide in the Unicellular Green Algae Cell Cycle: How Does It Work?

The regulatory role of some reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydrogen peroxide or nitric oxide, has been demonstrated in some higher plants and algae. Their involvement in regulation of the organism, tissue and single cell development can also be seen in many animals. In green cells, the redox potential is an important photosynthesis regulatory factor that may lead to an increase or decrease in growth rate. ROS and RNS are important signals involved in the regulation of photoautotrophic growth that, in turn, allow the cell to attain the commitment competence. Both hydrogen peroxide and nitric oxide are directly involved in algal cell development as the signals that regulate expression of proteins required for completing the cell cycle, such as cyclins and cyclin-dependent kinases, or histone proteins and E2F complex proteins. Such regulation seems to relate to the direct interaction of these signaling molecules with the redox-sensitive transcription factors, but also with regulation of signaling pathways including MAPK, G-protein and calmodulin-dependent pathways. In this paper, we aim to elucidate the involvement of hydrogen peroxide and nitric oxide in algal cell cycle regulation, considering the role of these molecules in higher plants. We also evaluate the commercial applicability of this knowledge. The creation of a simple tool, such as a precisely established modification of hydrogen peroxide and/or nitric oxide at the cellular level, leading to changes in the ROS-RNS cross-talk network, can be used for the optimization of the efficiency of algal cell growth and may be especially important in the context of increasing the role of algal biomass in science and industry. It could be a part of an important scientific challenge that biotechnology is currently focused on.

EPIP-Evoked Modifications of Redox, Lipid, and Pectin Homeostasis in the Abscission Zone of Lupine Flowers.

Yellow lupine is a great model for abscission-related research given that excessive flower abortion reduces its yield. It has been previously shown that the EPIP peptide, a fragment of LlIDL (INFLORESCENCE DEFICIENT IN ABSCISSION) amino-acid sequence, is a sufficient molecule to induce flower abortion, however, the question remains: What are the exact changes evoked by this peptide locally in abscission zone (AZ) cells? Therefore, we used EPIP peptide to monitor specific modifications accompanied by early steps of flower abscission directly in the AZ. EPIP stimulates the downstream elements of the pathway-HAESA and MITOGEN-ACTIVATED PROTEIN KINASE6 and induces cellular symptoms indicating AZ activation. The EPIP treatment disrupts redox homeostasis, involving the accumulation of H2O2 and upregulation of the enzymatic antioxidant system including superoxide dismutase, catalase, and ascorbate peroxidase. A weakening of the cell wall structure in response to EPIP is reflected by pectin demethylation, while a changing pattern of fatty acids and acyl lipids composition suggests a modification of lipid metabolism. Notably, the formation of a signaling molecule-phosphatidic acid is induced locally in EPIP-treated AZ. Collectively, all these changes indicate the switching of several metabolic and signaling pathways directly in the AZ in response to EPIP, which inevitably leads to flower abscission.

Does diclofenac act like a photosynthetic herbicide on green algae? Chlamydomonas reinhardtii synchronous culture-based study with atrazine as reference.

The non-steroidal anti-inflammatory drug diclofenac (DCF) is one of the commonly used and frequently detected drugs in water bodies, and several studies indicate its toxic effect on plants and algae. Studies performed with asynchronous Chlamydomonas reinhardtii cultures indicated that DCF inhibit the growth of population of the algae. Here, a synchronous population of C. reinhardtii, in which all cells are in the same developmental phase, is used. Following changes in cells size, photosynthetic activity and gene expression, we could compare, at the level of single cell, DCF-mediated effects with the effects caused by atrazine, a triazine herbicide that inhibits photosynthesis and triggers oxidative stress. Application of DCF and atrazine at the beginning of the cell cycle allowed us to follow the changes occurring in the cells in the subsequent stages of their development. Synchronized Chlamydomonas reinhardtii cultures (strain CC-1690, wild type) were exposed to diclofenac sodium salt (135 mg/L) or atrazine (77.6 µg/L). The cell suspension was sampled hourly (0-10 h) in the light period of the cell cycle to determine cell number and volume, photosynthetic pigment content, chlorophyll a fluorescence (OJIP test) in vivo, and selected gene expression (real-time qPCR), namely psbA, psaA, FSD1, MSD3 and APX1. The two toxicants differently influenced C. reinhardtii cells. Both substances decreased photosynthetic "vitality" (PI - performance index) of the cells, albeit for different reasons. While atrazine significantly disrupted the photosynthetic electron transport, resulting in excessive production of reactive oxygen species (ROS) and limited cell growth, DCF caused silencing of photosystem II (PSII) reaction centers, transforming them into "heat sinks", thus preventing significant ROS overproduction. Oxidative stress caused by atrazine was the probable reason for the rapid appearance of phytotoxic action soon after entering the cells, while the effects of DCF could only be seen several hours after treatment. A comparison of DCF-caused effects with the effects caused by atrazine led us to conclude that, although DCF cannot be regarded as typical photosynthetic herbicide, it exhibits an algicidal activity and can be potentially dangerous for aquatic plants and algae.

Diclofenac and atrazine restrict the growth of a synchronous Chlamydomonas reinhardtii population via various mechanisms.

Non-steroidal anti-inflammatory drug diclofenac (DCF) is commonly found in freshwater bodies and can have adverse effects on non-target organisms. Among the studies on DCF toxicity, several ones have reported its harmful effects on plants and algae. To gain a better understanding of the mechanisms of DCF toxicity towards green algae, we used a synchronous Chlamydomonas reinhardtii cc-1690 culture and compared DCF (135 mg/L) effects with effects caused by atrazine (ATR; 77.6 µg/L), an herbicide with a well-known mechanism of toxic action. To achieve our goal, cell number and size, photosynthetic oxygen consumption/evolution, chlorophyll a fluorescence in vivo, H2O2 production by the cells, antioxidative enzymes encoding genes expression were analyzed during light phase of the cell cycle. We have found, that DCF and ATR affect C. reinhardtii through different mechanisms. ATR inhibited the photosynthetic electron transport chain and induced oxidative stress in chloroplast. Such chloroplastic energetics disruption indirectly influenced respiration, the intensification of which could partially mitigate low efficiency of photosynthetic energy production. As a result, ATR inhibited the growth of single cell leading to limitation in C. reinhardtii population development. In contrast to ATR-treated algae, in DCF-treated cells the fraction of active PSII reaction centers was diminished without drastic changes in electron transport or oxidative stress symptoms in chloroplast. However, significant increase in transcript level of gene encoding for mitochondria-located catalase indicates respiratory processes as a source of H2O2 overproduced in the DCF-treated cells. Because the single cell growth was not strongly affected by DCF, its adverse effect on progeny cell number seemed to be related rather to arresting of cell divisions. Concluding, although the DCF phytotoxic action appeared to be different from the action of the typical herbicide ATR, it can act as algal growth-inhibiting factor in the environment.

Disruption of the Auxin Gradient in the Abscission Zone Area Evokes Asymmetrical Changes Leading to Flower Separation in Yellow Lupine.

How auxin transport regulates organ abscission is a long-standing and intriguing question. Polar auxin transport across the abscission zone (AZ) plays a more important role in the regulation of abscission than a local concentration of this hormone. We recently reported the existence of a spatiotemporal sequential pattern of the indole-3-acetic acid (IAA) localization in the area of the yellow lupine AZ, which is a place of flower detachment. In this study, we performed analyses of AZ following treatment with an inhibitor of polar auxin transport (2,3,5-triiodobenzoic acid (TIBA)). Once we applied TIBA directly onto the AZ, we observed a strong response as demonstrated by enhanced flower abscission. To elucidate the molecular events caused by the inhibition of auxin movement, we divided the AZ into the distal and proximal part. TIBA triggered the formation of the IAA gradient between these two parts. The AZ-marker genes, which encode the downstream molecular components of the inflorescence deficient in abscission (IDA)-signaling system executing the abscission, were expressed in the distal part. The accumulation of IAA in the proximal area accelerated the biosynthesis of abscisic acid and ethylene (stimulators of flower separation), which was also reflected at the transcriptional level. Accumulated IAA up-regulated reactive oxygen species (ROS) detoxification mechanisms. Collectively, we provide new information regarding auxin-regulated processes operating in specific areas of the AZ.

Exogenously applied hydrogen peroxide modifies the course of the Chlamydomonas reinhardtii cell cycle.

The interaction of NO and H2O2 in the regulation of plant development is well documented. We have recently shown that the content of NO and H2O2 changes in a characteristic way during the cell cycle of Chlamydomonas reinhardtii (Pokora et al., 2017), which implies participation of these molecules in the regulation of Chlamydomonas development. To verify this assumption, H2O2 was supplied at a concentration about 1.5 times higher than that determined in the control cells. Cells were synchronized by alternating the light/dark (10/14 h) regimen. H2O2 was added to zoospore suspensions, previously held in the dark, and cells growing for 3, 6, and 9 h in the light. The data indicate that, depending on the phase of the Chlamydomonas cell cycle, H2O2, via mild modification of redox homeostasis, may: a) accelerate or delay the duration of the cell cycle; b) increase the number of replication rounds occurring in one cell cycle; c) modify the biomass and cell volume of progeny cells and d) accelerate the liberation of daughter cells. This provides a tool to control the development of Chlamydomonas cell and thus offers the opportunity to obtain a population of cells with characteristics desired in biotechnology.

Changes in nitric oxide/hydrogen peroxide content and cell cycle progression: Study with synchronized cultures of green alga Chlamydomonas reinhardtii.

The present study aimed to evaluate the possible relationship between the changes in hydrogen peroxide (H2O2) and nitric oxide (NO) content and the course of growth and reproductive processes of the cell cycle of Chlamydomonas reinhardtii. The peak of H2O2 observed at the beginning of the cell cycle was found to originate from Fe-SOD and Mn-SODchl. activity and result from the alternation in the photosynthetic processes caused by the dark-to-light transition of daughter cells. A rapid increase in NO concentration, observed before the light-to-dark cell transition, originated from NR and NIR activity and was followed by a photosynthesis-independent, Mn-SODchl.-mediated increases in H2O2 production. This H2O2 peak overlapped the beginning of Chlamydomonas cell division, which was indicated by a profile of CYCs and CDKs characteristic of cells' passage through the G1/S and S/M checkpoints. Taken together, our results show that there is a clear relationship between the course of the Chlamydomonas cell cycle and typical changes in the H2O2/NO ratio, as well as changes in expression and activity of enzymes involved in generation and scavenging of these signaling molecules.

Effects of juglone and lawsone on oxidative stress in maize coleoptile cells treated with IAA.

Naphthoquinones are secondary metabolites widely distributed in nature and produced by bacteria, fungi and higher plants. Their biological activity may result from induction of oxidative stress, caused by redox cycling or direct interaction with cellular macromolecules, in which quinones act as electrophiles. The redox homeostasis is known as one of factors involved in auxin-mediated plant growth regulation. To date, however, little is known about the crosstalk between reactive oxygen species (ROS) produced by quinones and the plant growth hormone auxin (IAA). In this study, redox cycling properties of two naphthoquinones, juglone (5-hydroxy-1,4-naphthoquinone) and lawsone (2-hydroxy-1,4-naphthoquinone), were compared in experiments performed on maize coleoptile segments incubated with or without the addition of IAA. It was found that lawsone was much more effective than juglone in increasing both H2O2 production and the activity of antioxidative enzymes (SOD, POX and CAT) in coleoptile cells, regardless of the presence of IAA. An increase in the activity of Cu/Zn-SOD isoenzymes induced by both naphthoquinones suggests that juglone- and lawsone-generated H2O2 was primarily produced in the cytosolic and cell wall spaces. The cell potential to neutralize hydrogen peroxide, determined by POX and CAT activity, pointed to activity of catalase as the main enzymatic mechanism responsible for degradation of H2O2 Therefore, we assumed that generation of H2O2, induced more efficiently by LW than JG, was the major factor accounting for differences in the toxicity of naphthoquinones in maize coleoptiles. The role of auxin in the process appeared negligible. Moreover, the results suggested that oxidative stress imposed by JG and LW was one of mechanisms of allelopathic action of the studied quinones in plants.

Time-dependent changes in antioxidative enzyme expression and photosynthetic activity of Chlamydomonas reinhardtii cells under acute exposure to cadmium and anthracene.

Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are present in the freshwater environment at concentrations that can be hazardous to the biota. Among HMs and PAHs, cadmium (Cd) and anthracene (ANT) are the most prevalent and toxic ones. The response of Chlamydomonas cells to Cd and ANT at concentrations that markedly reduced the growth of algal population was investigated in this study. At such concentrations, both cadmium and anthracene were recognized as oxidative stress inducers, since high concentration of H2O2 in treated cultures was observed. Therefore, as a part of the "molecular phase" of the cell response to this stress, we examined the time-dependent expression of genes encoding the main antioxidative enzymes: superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as the activity of these enzymes in cells, with special attention paid to chloroplastic and mitochondrial isoforms of SOD. To characterize the cell response at the "physiological level", we examined the photosynthetic activity of stressed cells via analysis of chlorophyll a fluorescence in vivo. In contrast to standard ecotoxicity studies in which the growth end-points are usually determined, herein we present time-dependent changes in algal cell response to Cd- and ANT-induced stress. The most significant effect(s) of the toxicants on photosynthetic activity was observed in the 6th hour, when strong depression of PI parameter value, an over 50 percent reduction of the active reaction center fraction (RC0) and a 3-fold increase in non-photochemical energy dissipation (DI0/RC) were noted. At the same time, the increase (up to 2.5-fold) in mRNA transcript of SOD and CAT genes, followed by the enhancement in the enzyme activity was observed. The high expression of the Msd 3 gene in treated Chlamydomonas cells probably complements the partial loss of chloroplast Fe-SOD and APX activity, while catalase and Mn-SOD 5 seem to be the major enzymes responsible for mitochondrion protection. The progressive increase in SOD and CAT activities seems to be involved in the recovery of photosynthesis within 12-24h after the application of the toxicants.

Adaptation strategies of two closely related Desmodesmus armatus (green alga) strains contained different amounts of cadmium: a study with light-induced synchronized cultures of algae.

During the Desmodesmus armatus cell cycle, 8-celled coenobia of 276-4d strain accumulated a much lower amounts of cadmium than unicells of B1-76 strain. Cadmium reduced growth and photosynthesis in the cells of strain B1-76, but not those of 276-4d strain. Cells of 276-4d strain revealed a higher activity of superoxide dismutase (SOD) isoforms, in particular the activity and protein content of Fe-SOD. Cu/Zn-SOD was earlier and much stronger induced by cadmium in 276-4d than in B1-76 strain, whereas Fe- and Mn-SOD activity and Fe-SOD synthesis were induced only in 276-4d strain. Cadmium did not affect the heat shock protein 70 synthesis in B1-76 strain, but significantly stimulated this process in 276-4d strain. The level of glutathione increased 30-fold during cell development of Cd-exposed 276-4d strain, while in B1-76 it increased about 12 timed. Matured cells of both strains exposed to cadmium produced comparable amounts of phytochelatins and other thiol peptides, but their production in young cells of B1-76 strain was much higher than in 276-4d strain. In conclusion, a complex of internal detoxification mechanisms appeared to be more efficient in cells of 276-4d strain than B1-76 one.

Induction time of Fe-SOD synthesis and activity determine different tolerance of two Desmodesmus (green algae) strains to chloridazon: a study with synchronized cultures.

Cells of two Desmodesmus armatus strains (276-4a and 276-4d) grown asynchronously in batch cultures after 24-h treatment revealed different tolerance to chloridazon (photosynthetic herbicide) applied at a concentration of 3.45 mg L(-1). To find time- and cell cycle-dependent biochemical reasons leading to such a difference, a population of young autospores of both strains synchronized by a light/dark (14/10) regime were exposed to chloridazon at the initiation of the light period. Chloridazon reduced the growth and number of divisions of cell strain 276-4d. In consequence, at the end of the dark phase the number of released autospores was reduced by 50% compared with the control. In contrast, the growth and reproductive processes of cell strains 276-4a was unaffected. Moreover, chloridazon treatment speeded up cell development, as a result of which the release of autospores took this process observed in the control cells over. There is a relationship between photosynthetic activity response to chloridazon and time-dependent changes in Fe-SOD content and activity. The energy trapped in the reaction centre (RC) was similar in both strains, but the amount of energy absorbed by RCs was twice as high in strain 276-4d as in 276-4a. In consequence, non-photochemical energy dissipation occurring in the cells of 276-4d strain far exceed the value obtained for 276-4a strain. The control cells of both strains differed significantly in the content of FSD 1 and FSD 2 proteins, whereas the differences in Fe-SOD isoforms activities were slight. The 8-fold increase in SOD content in CHD treated cells of strain 276-4a was associated with the transience of photosynthetic efficiency impairment. In CHD treated cells of strain 276-4d, neither activity of Fe-SOD nor FSDs protein content was instantly affected. Different response of developing cells of two Desmodesmus strains to CHD is influenced by the inherent features of cells; the short time required to induce stress adaptive mechanism involving chloroplastic Fe-SOD activity and biosynthesis in the cells of CHD tolerant strain 276-4a seems to play the key role, being an overriding on the high, but not induced in response to stress, FSD protein level and activity in cells of strain 276-4d.

The combined effect of anthracene and cadmium on photosynthetic activity of three Desmodesmus (Chlorophyta) species.

Individual toxicity of heavy metals (HM) and polycyclic aromatic hydrocarbons (PAH) to plants living in water bodies is well-documented. In view of frequent joint occurrence of these compounds in the environment, plants are subjected to damage from their combined action. Cadmium and anthracene can generate production of reactive oxygen species (ROS). We have recently detected elevated activity of Fe- and Mn-SOD isoforms, indicating chloroplast and mitochondrion as the main sites of combined toxicity of HM and PAH. In the present paper, short-term (1-24 h) experiments on the mechanism of combined toxicity of anthracene and cadmium to the photosynthesis of three Desmodesmus species are reported. Inhibition, stimulation or no effect on the oxygen evolution was observed following the treatment with the contaminants when applied either separately or jointly. The response pattern was both strongly species- and time-dependent. In contrast, the photosynthetic activity of cells, expressed by chlorophyll fluorescence parameters, was substantially unaffected, since no effect or, in several cases, a slight stimulation of PS II quantum efficiency (Phi PS II) were noted. A characteristic relationship between the SOD activity and the qN values was observed. The treatment of Desmodesmus cells with anthracene or cadmium had either no effect or slightly enhanced either the SOD activity or the qN value, whereas the mixture of the contaminants resulted in a multifold increase in both the SOD activity and the qN values. The results suggest that chloroplasts of algae are well protected against the combined action of the two contaminants the toxicity of which should be attributed to nucleocytoplasmic compartments and reproductive processes of the cell cycle.