Cyanobacterial VOCs ß-ionone and ß-cyclocitral poisoning Lemna turionifera by triggering programmed cell death.

ß-Ionone and ß-cyclocitral are two typical components in cyanobacterial volatiles, which can poison aquatic plants and even cause death. To reveal the toxic mechanisms of the two compounds on aquatic plants through programmed cell death (PCD), the photosynthetic capacities, caspase-3-like activity, DNA fragmentation and ladders, as well as expression of the genes associated with PCD in Lemna turionifera were investigated in exposure to ß-ionone (0.2 mM) and ß-cyclocitral (0.4 mM) at lethal concentration. With prolonging the treatment time, L. turionifera fronds gradually died, and photosynthetic capacities gradually reduced and even disappeared at the 96th h. This demonstrated that the death process might be a PCD rather than a necrosis, due to the gradual loss of physiological activities. When L. turionifera underwent the death, caspase-3-like was activated after 3 h, and reached to the strongest activity at the 24th h. TUNEL-positive nuclei were detected after 12 h, and appeared in large numbers at the 48th h. The DNA was cleaved by Ca2+-dependent endonucleases and showed obviously ladders. In addition, the expression of 5 genes (TSPO, ERN1, CTSB, CYC, and ATR) positively related with PCD initiation was up-regulated, while the expression of 2 genes (RRM2 and TUBA) negatively related with PCD initiation was down-regulated. Therefore, ß-ionone and ß-cyclocitral can poison L. turionifera by adjusting related gene expression to trigger PCD.

Identification of algicidal monoterpenoids from four chemotypes of Cinnamomum camphora and their algicidal mechanisms on Microcystis aeruginosa.

Cyanobacterial blooms cause serious environmental issues, and plant secondary metabolites are considered as new algaecide for controlling them. Cinnamomum camphora produces a wide spectrum of terpenoids and has 4 main chemotypes, including linalool, camphor, eucalyptol and borneol chemotype. To develop the new cyanobacterial algaecide by using suitable chemotype of Cinnamomum camphora and the main terpenoids, we analyzed the terpenoid composition in the 4 chemotype extracts, evaluated the algicidal effects of the extracts and their typical monoterpenoids on Microcystis aeruginosa, and investigated the algicidal mechanism of the stronger algicidal agents. Among the 4 chemotypes, eucalyptol and borneol chemotype extracts exhibited stronger algicidal effects. In the 4 chemotype extracts, monoterpenoids were the main compounds, of which linalool, camphor, eucalyptol and borneol were the typical components. Among the 4 typical monoterpenoids, eucalyptol and borneol showed stronger algicidal effects, which killed 78.8% and 100% M. aeruginosa cells, respectively, at 1.2 mM after 48 h. In 1.2 mM eucalyptol and borneol treatments, the reactive oxygen species levels markedly increased, and the caspase-3-like activity also raised. With prolonging the treatment time, M. aeruginosa cells gradually shrank and wrinkled, and the cell TUNEL fluorescence intensity and DNA degradation gradually enhanced, indicating that the lethal mechanism is causing apoptosis-like programmed cell death (PCD). Therefore, eucalyptol and borneol chemotype extracts and their typical monoterpenoids have the potential for developing as algaecides to control cyanobacteria through triggering apoptosis-like PCD.

Adjusting function of camphor on primary metabolism in Cinnamomum camphora stressed by high temperature.

Cinnamomum camphora has great economic value for its wide utilization in traditional medicine and furniture material, and releases lots of monoterpenes to tolerate high temperature. To uncover the adjusting function of monoterpenes on primary metabolism and promoting their utilization as anti-high temperature agents, the photosynthetic capacities, primary metabolite levels, cell ultrastructure and associated gene expression were surveyed in C. camphora when it was blocked monoterpene biosynthesis with fosmidomycin (Fos) and fumigated with camphor (a typical monoterpene in the plant) under high temperature (Fos+38 °C+camphor). Compared with the control (28 °C), high temperature at 38 °C decreased the starch content and starch grain size, and increased the fructose, glucose, sucrose and soluble sugar content. Meanwhile, high temperature also raised the lipid content, with the increase of lipid droplet size and numbers. These variations were further intensified in Fos+ 38 °C treatment. Compared with Fos+ 38 °C treatment, Fos+ 38 °C+camphor treatment improved the starch accumulation by promoting 4 gene expression in starch biosynthesis, and lowered the sugar content by suppressing 3 gene expression in pentose phosphate pathway and promoting 15 gene expression in glycolysis and tricarboxylic acid cycle. Meanwhile, Fos+ 38 °C+camphor treatment also lowered the lipid content, which may be caused by the down-regulation of 2 genes in fatty acid formation and up-regulation of 4 genes in fatty acid decomposition. Although Fos+ 38 °C+camphor treatment improved the photosynthetic capacities in contrast to Fos+ 38 °C treatment, it cannot explain the variations of these primary metabolite levels. Therefore, camphor should adjust related gene expression to maintain the primary metabolism in C. camphora tolerating high temperature.

Acetylcholine (ACh) enhances Cd tolerance through transporting ACh in vesicles and modifying Cd absorption in duckweed (Lemna turionifera 5511).

Acetylcholine (ACh), an important neurotransmitter, plays a role in resistance to abiotic stress. However, the role of ACh during cadmium (Cd) resistance in duckweed (Lemna turionifera 5511) remains uncharacterized. In this study, the changes of endogenous ACh in duckweed under Cd stress has been investigated. Also, how exogenous ACh affects duckweed's ability to withstand Cd stress was studied. The ACh sensor transgenic duckweed (ACh 3.0) showed the ACh signal response under Cd stress. And ACh was wrapped and released in vesicles. Cd stress promoted ACh content in duckweed. The gene expression analysis showed an improved fatty acid metabolism and choline transport. Moreover, exogenous ACh addition enhanced Cd tolerance and decreased Cd accumulation in duckweed. ACh supplement reduced the root abscission rate, alleviated leaf etiolation, and improved chlorophyll fluorescence parameters under Cd stress. A modified calcium (Ca2+) flux and improved Cd2+ absorption were present in conjunction with it. Thus, we speculate that ACh could improve Cd resistance by promoting the uptake and accumulation of Cd, as well as the response of the Ca2+ signaling pathway. Also, plant-derived extracellular vesicles (PDEVs) were extracted during Cd stress. Therefore, these results provide new insights into the response of ACh during Cd stress.

Thermal protection function of camphor on Cinnamomum camphora cell membrane by acting as a signaling molecule.

Isoprenoids serve important functions in protecting plant membranes against high temperature. Cinnamomum camphora is an excellent economic tree species, and releases plenty of monoterpenes. To uncover the protective mechanism of monoterpenes on the membrane system for promoting their development and utilization as anti-high temperature agents, the membrane permeability, cell ultrastructure, membrane lipid variations and related gene expression were investigated in C. camphora fumigated with camphor, one of the main monoterpenes in the plant, after fosmidomycin (Fos) blocking the monoterpene biosynthesis under high temperature (Fos+38 °C + C). High temperature at 38 °C caused the rupture of plasma as well as chloroplast and mitochondrion membranes, deformation of chloroplasts and mitochondria, and electrolyte leakage in C. camphora. High temperature with Fos treatment (Fos+38 °C) aggravated the damage, while camphor fumigation (Fos+38 °C + C) showed alleviating effects. High temperature at 38 °C disturbed the membrane lipid equilibrium by reducing the levels of 14 phosphatidylcholine, 8 phosphatidylglycerol and 6 phosphatidylethanolamine molecules, and increasing the levels of 8 phosphatidic acid, 4 diacylglycerol, 5 phosphatidylinositol, 16 sphingomyelin and 5 ceramide phosphoethanolamine molecules. Fos+38 °C treatment primarily exhibited intensifying effects on the disturbance, while these membrane lipid levels in Fos+38 °C + C5 (5 µM camphor) treatment exhibited variation tendencies to the control at 28 °C. This should result from the expression alterations of the genes related with phospholipid biosynthesis, fatty acid metabolism, and sphingolipid metabolism. It can be speculated that camphor can maintain membrane lipid stabilization in C. camphora under high temperature by acting as a signaling molecule.

Emission of cyanobacterial volatile organic compounds and their roles in blooms.

Cyanobacteria are photosynthetic prokaryotes and one of dominant species in eutrophicated waters, which easily burst blooms in summer with high irradiance and temperature conditions. In response to high irradiance, high temperature, and nutrient conditions, cyanobacteria release abundant of volatile organic compounds (VOCs) by up-regulating related gene expression and oxidatively degrading ß-carotene. These VOCs not only increase offensive odor in waters, but also transfer allelopathic signals to algae and aquatic plants, resulting in cyanobacteria dominating eutrophicated waters. Among these VOCs, ß-cyclocitral, α-ionone, ß-ionone, limonene, longifolene, and eucalyptol have been identified as the main allelopathic agents, which even directly kill algae by inducing programmed cell death (PCD). The VOCs released from cyanobacteria, especially the ruptured cells, exhibit repelling effects on the herbivores, which is beneficial to survival of the population. Cyanobacterial VOCs might transfer aggregating information among homogeneous species, so the acceptors initiate aggregation to resist the coming stresses. It can be speculated that the adverse conditions can promote VOC emission from cyanobacteria, which play important roles in cyanobacteria dominating eutrophicated waters and even bursting blooms.

Age effects of Moso bamboo on leaf isoprene emission characteristics.

Isoprene is a highly reactive volatile organic compound that significantly affects atmospheric oxidant capacity, regional air quality, and climate change. Moso bamboo (Phyllostachys edulis), a species widely distributed in tropical and subtropical regions, particularly in China, is a strong isoprene emitter with great potential for carbon sequestration. Carbon sequestration is negatively correlated with culm age; however, the effect of this correlation on isoprene emissions remains unknown. In this study, we investigated the photosynthetic and isoprene emission characteristics of Moso bamboo at different culm ages. The results showed that the age effect on isoprene emission was different from that on photosynthesis; the net photosynthesis rate (Pn) was the highest in young, followed by mature, and then old bamboo, whereas the isoprene emission rate (Iso) was the highest in young, followed by old, and then mature bamboo. Moreover, the percentage of carbon loss as isoprene emission (C-loss) during photosynthesis of old bamboo was 35% higher than that of mature bamboo under standard conditions (leaf temperature: 30°C; light intensity: 1000 µmol m-2 s-1). Therefore, we strongly recommend considering the culm age when establishing an isoprene emission model of Moso bamboo. Additionally, because the Iso and C-loss of old bamboo were higher than those of mature bamboo, we suggest that attention should be paid to the management of bamboo age structure and timely felling of aged bamboo to reduce environmental risk.

Toxic mechanism of two cyanobacterial volatiles ß-cyclocitral and ß-ionone on the photosynthesis in duckweed by altering gene expression.

Volatile organic compounds (VOCs) promote cyanobacteria dominating eutrophicated waters, with aquatic plant decrease and even disappearance. To uncover the toxic mechanism of cyanobacterial VOCs on aquatic plants, we investigated the growth, photosynthetic pigment levels, photosynthetic abilities and related gene expression in duckweed treated with ß-cyclocitral and ß-ionone, 2 main components in the VOCs. The levels of chlorophylls and carotenoids gradually declined with raising the concentration of the 2 compounds and prolonging the treatment time. Their decline should result from the down-regulation of 8 genes associated with photosynthetic pigment biosynthesis and up-regulation of 2 genes involved in carotenoid degradation. The reduction was also found in the photosystem II (PSII) efficiency and O2 evolution rate, which should result from the lowered photosynthetic pigment levels and down-regulation of 38 genes related with photosynthetic process. The frond numbers, total frond area and fresh weight gradually decreased with raising the 2 compound concentration, which may result from the lowered photosynthetic abilities as well as down-regulated expression of 7 genes associated with growth-promoting hormone biosynthesis and signal transduction. It can be speculated that cyanobacterial VOCs may poison aquatic plants by lowering the photosynthesis and growth through altering related gene expression.

Astaxanthin accumulation in Microcystis aeruginosa under different light quality.

The aim of this work was to uncover the astaxanthin biosynthesis mechanism in Microcystis aeruginosa under optimum light quality, and promote astaxanthin production using this alga. Among purple, blue and red light, only purple light promoted M. aeruginosa cell growth compared with white light, due to up-regulating expression of the genes related with DNA replication. An increase was detected in the photosynthetic rate under purple light, which should be caused by the raised carotenoid content and up-regulation of the genes associated with light reaction and carbon fixation. Compared with white light, purple light increased the levels of ß-carotene, zeaxanthin and astaxanthin by up-regulating expression of the genes related with methylerythritol-4-phosphate pathway (MEP) and astaxanthin biosynthesis. For red and blue light, they did not impact or declined the content of astaxanthin and its precursors. Therefore, purple light promoted M. aeruginosa cell growth and astaxanthin production by up-regulating related gene expression.

The uppermost monoterpenes improving Cinnamomum camphora thermotolerance by serving signaling functions.

Terpenes serve important functions in enhancing plant thermotolerance. Cinnamomum camphora mainly has eucalyptol (EuL), camphor (CmR), linalool (LnL) and borneol (BeL) chemotypes basing on the uppermost monoterpenes. To reveal the thermotolerance mechanisms of these uppermost monoterpenes (eucalyptol, camphor, linalool, and borneol) in C. camphora, we surveyed the ROS metabolism and photosynthesis in the 4 chemotypes fumigated with the corresponding uppermost monoterpene after fosmidomycin (Fos) inhibiting monoterpene synthesis under high temperature at 38°C (Fos+38°C+monoterpene), and investigated the related gene expression in EuL and CmR. Meanwhile, the thermotolerance differences among the 4 uppermost monoterpenes were analyzed. In contrast to normal temperature (28°C), ROS levels and antioxidant enzyme activities in the 4 chemotypes increased under 38°C, and further increased in the treatment with Fos inhibiting monoterpene synthesis at 38°C (Fos+38°C), which may be caused by the alterations in expression of the genes related with non-enzymatic and enzymatic antioxidant formation according to the analyses in EuL and CmR. Compared with Fos+38°C treatment, Fos+38°C+monoterpene treatments lowered ROS levels and antioxidant enzyme activities for the increased non-enzymatic antioxidant gene expression and decreased enzymatic antioxidant gene expression, respectively. High temperature at 38°C reduced the chlorophyll and carotenoid content as well as photosynthetic abilities, which may result from the declined expression of the genes associated with photosynthetic pigment biosynthesis, light reaction, and carbon fixation. Fos+38°C treatment aggravated the reduction. In contrast to Fos+38°C treatment, Fos+38°C+monoterpene treatments increased photosynthetic pigment content and improved photosynthetic abilities by up-regulating related gene expression. Among the 4 uppermost monoterpenes, camphor showed strong abilities in lowering ROS and maintaining photosynthesis, while eucalyptol showed weak abilities. This was consistent with the recovery effects of the gene expression in the treatments with camphor and eucalyptol fumigation. Therefore, the uppermost monoterpenes can enhance C. camphora thermotolerance as signaling molecules, and may have differences in the signaling functions.

Programmed cell death of Chlamydomonas reinhardtii induced by three cyanobacterial volatiles ß-ionone, limonene and longifolene.

ß-Ionone, limonene and longifolene are 3 main components in cyanobacterial volatile organic compounds, which are formed through different pathways and can poison and even kill other algae. To uncover their toxic mechanism from programmed cell death (PCD), the photosynthetic pigments, chlorophyll fluorescence, caspase-like activities, cell size, nuclear variations and DNA ladders were investigated in Chlamydomonas reinhardtii treated with ß-ionone (0.2 mM), limonene (0.2 mM) and longifolene (0.4 mM) at lethal concentration during 24 h. In the treatments with the 3 compounds, the photosynthetic pigments in C. reinhardtii cells gradually degraded, and Fv/Fm gradually decreased and disappeared at 24 h, suggesting that the cell death might be a PCD, due to the physiological activities gradually disappearing. During the cell death, the activities of caspase-9-like and caspase-3-like significantly increased, with the highest at 1 h. With prolonging the treatment time, C. reinhardtii cells gradually shrank, and the nuclei concentrated firstly following by a broken process, with moving to the cell edge. For DNA, obvious ladders were detected at 1 h, and then they gradually degraded to fragments of 100-250 bp at 24 h. These hallmarks suggested that ß-ionone, limonene and longifolene may poison other algae by inducing PCD.

Optimization of the nitrogen and oxygen element distribution in microalgae by ammonia torrefaction pretreatment and subsequent fast pyrolysis process for the production of N-containing chemicals.

In this work, ammonia (NH3) torrefaction pretreatment (ATP) was developed to optimize the nitrogen and oxygen element distribution of microalgae via the N-doping and oxygen removal reaction, which could obviously improve the potential use of microalgae as a feedstock for the production of N-heterocyclic chemicals through fast pyrolysis technology. The nitrogen content increased from 8.3% of raw microalgae to 11.51% at 300 °C of ATP, while the oxygen content decreased from 35.96% to 21.61%, because of the Maillard reactions. In addition, the nitrogen-doping ratio and oxygen removal ratio of ATP was much higher than the conventional nitrogen torrefaction pretreatment (NTP). With the increase of ATP torrefaction temperature or the pyrolysis temperature, the relative content of the N-containing compounds increased, while the O-containing compounds decreased. For the N-heterocyclic chemicals, higher pyrolysis temperature favored the formation of pyrroles, while inhibited the formation of pyridines and indoles.

Effects of high light and temperature on Microcystis aeruginosa cell growth and ß-cyclocitral emission.

Cyanobacteria always massively grow and even occur blooms in summer, with releasing amount of ß-cyclocitral. To uncover the effects of summer high irradiance and temperature on cyanobacterial growth and ß-cyclocitral emission, the cell growth, reactive oxygen species (ROS) levels, photosynthetic pigment content, chlorophyll fluorescence and ß-cyclocitral emission were investigated in Microcystis aeruginosa under high light and temperature. Compared to the control under 50 µmol m-2·s-1, the cell growth was promoted under 100 µmol m-2·s-1, but inhibited under 500 and 1000 µmol m-2·s-1. The inhibition was also detected under high temperature at 30 and 35 °C in contrast to the control at 25 °C. Under high light and high temperature, M. aeruginosa increased ROS levels and reduced photosynthetic pigment content and photosystem II (PSII) efficiency, which resulted in the inhibition on cell growth. With increasing the light intensity and temperature, 1O2 levels gradually increased, while ß-carotene content gradually decreased by quenching 1O2, with increasing ß-cyclocitral emission. In summer, high irradiance and temperature not benefited the growth of cyanobacteria, but the emission of ß-cyclocitral derived from ß-carotene quenching 1O2 may offset the disadvantages by poisoning other algae.

Toxic mechanism of eucalyptol and ß-cyclocitral on Chlamydomonas reinhardtii by inducing programmed cell death.

Eucalyptol and ß-cyclocitral are 2 main compounds in cyanobacterial volatile organic compounds and can poison other algae. To uncover the toxic mechanism of the 2 compounds, the cell growth, photosynthetic abilities, H2O2 production, caspase-like activities, nuclear variation and DNA laddering were investigated in Chlamydomonas reinhardtii treated with eucalyptol and ß-cyclocitral. Eucalyptol at ≥ 0.1 mM and ß-cyclocitral at ≥ 0.05 mM showed toxic effects on C. reinhardtii cells, and 1.2 mM eucalyptol and 0.4 mM ß-cyclocitral killed the whole of the cells during 24 h. During the death process, the photosynthetic pigment gradually degraded, and Fv/Fm gradually declined, indicating that the death is not a necrosis due to the gradual disappearance of the physiological process. In the treatments with 1.2 mM eucalyptol and 0.4 mM ß-cyclocitral, H2O2 content burst at 10 min and 30 min, respectively. Caspase-9-like and caspase-3-like were activated, and cell nucleuses concentrated firstly and then broke with prolonging the treatment time. Meanwhile, DNA showed laddering after 1 h, and was gradually cleaved by Ca2+-dependent endonucleases to mainly about 100-250 bp fragments. These hallmarks indicated that eucalyptol and ß-cyclocitral may poison other algal cells by inducing programmed cell death triggered by the increased H2O2.

Declined cadmium accumulation in Na+/H+ antiporter (NHX1) transgenic duckweed under cadmium stress.

Cadmium (Cd) is a serious threat to plants health. Though some genes have been reported to get involved in the regulation of tolerance to Cd, the mechanisms underlying this process are not fully understood. Na+/H+ antiporter (NHX1) plays an important role in Na+/H+ trafficking. The salt and cadmium stress tolerance were found to be enhanced by NHX1 in duckweed according to our previous study, however, its function in Cd2+ flux under Cd stress has not been studied. Here we explored the Cd2+ flux in wild type (WT) and NHX1 transgenic duckweed (NHX1) under Cd stress. We found that the Cd2+ influx in NHX1 duckweed was significantly declined, followed by an increased Cd2+ efflux after 20 min treatment of Cd, which resulted a less accumulation of Cd in NHX1. Reversely, inhibition of NHX1 by amiloride treatment, enhanced Cd2+ influx in NHX1 duckweed, subsequently delayed Cd2+ efflux in both genotypes of duckweed under Cd2+ shock. H+ efflux in NHX1 duckweed was lower compare with that in WT with 20 min Cd2+ shock. NHX1 also increased the pH value with Cd2+ stress in the transgenic rhizoid. These finding suggested a new function of NHX1 in regulation of Cd2+ and H+ flow during short-term Cd2+ shock.

Why Algae Release Volatile Organic Compounds-The Emission and Roles.

A wide spectrum of volatile organic compounds (VOCs) are released from algae in aquatic ecosystems. Environmental factors such as light, temperature, nutrition conditions and abiotic stresses affect their emission. These VOCs can enhance the resistance to abiotic stresses, transfer information between algae, play allelopathic roles, and protect against predators. For homogeneous algae, the VOCs released from algal cells under stress conditions transfer stress information to other cells, and induce the acceptors to make a preparation for the upcoming stresses. For heterogeneous algae and aquatic macrophytes, the VOCs show allelopathic effects on the heterogeneous neighbors, which benefit to the emitter growth and competing for nutrients. In cyanobacterial VOCs, some compounds such as limonene, eucalyptol, ß-cyclocitral, α-ionone, ß-ionone and geranylacetone have been detected as the allelopathic agents. In addition, VOCs can protect the emitters from predation by predators. It can be speculated that the emission of VOCs is critical for algae coping with the complicated and changeable aquatic ecosystems.

Isoprene Acts as a Signaling Molecule in Gene Networks Important for Stress Responses and Plant Growth.

Isoprene synthase converts dimethylallyl diphosphate to isoprene and appears to be necessary and sufficient to allow plants to emit isoprene at significant rates. Isoprene can protect plants from abiotic stress but is not produced naturally by all plants; for example, Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) do not produce isoprene. It is typically present at very low concentrations, suggesting a role as a signaling molecule; however, its exact physiological role and mechanism of action are not fully understood. We transformed Arabidopsis with a Eucalyptus globulus isoprene synthase The regulatory mechanisms of photosynthesis and isoprene emission were similar to those of native emitters, indicating that regulation of isoprene emission is not specific to isoprene-emitting species. Leaf chlorophyll and carotenoid contents were enhanced by isoprene, which also had a marked positive effect on hypocotyl, cotyledon, leaf, and inflorescence growth in Arabidopsis. By contrast, leaf and stem growth was reduced in tobacco engineered to emit isoprene. Expression of genes belonging to signaling networks or associated with specific growth regulators (e.g. gibberellic acid that promotes growth and jasmonic acid that promotes defense) and genes that lead to stress tolerance was altered by isoprene emission. Isoprene likely executes its effects on growth and stress tolerance through direct regulation of gene expression. Enhancement of jasmonic acid-mediated defense signaling by isoprene may trigger a growth-defense tradeoff leading to variations in the growth response. Our data support a role for isoprene as a signaling molecule.

Linalool- and α-terpineol-induced programmed cell death in Chlamydomonas reinhardtii.

Plant allelochemicals effectively inhibit and/ or control algal growth, and have potential to use as algaecide. To uncover the lethal mechanism of 2 anti-algal compounds linalool and α-terpineol identified from Cinnamomum camphora extracts, and promote their development as algaecide, the H2O2 production, photosynthetic abilities, caspase-like activities, nuclear changes and DNA degradation were investigated in Chlamydomonas reinhardtii treated with the 2 compounds. H2O2 content burst in linalool treatment at 0.5 h and in α-terpineol treatment at 1 h, with increases of 2.7 folds and 1.3 folds, respectively, compared to that at 0 h. The photosynthetic pigments gradually degraded, and Fv/Fm gradually declined to zero, indicating that the cell death was not a necrosis due to the gradual disappearance of physiological process. In C. reinhardtii cells, the caspase-9-like and caspase-3-like were activated in the treatments with the 2 compounds for 1 h. With prolonging the treatment time, the fluorescent intensity of the cell nucleuses stained by DAPI gradually enhanced and then faded, and the genomic DNA isolated from the cells gradually degraded. These hallmarks indicated that the death of C. reinhardtii cells in linalool and α-terpineol treatments was a programmed cell death (PCD) triggered by the increased reactive oxygen species (ROS). Compared to α-terpineol treatment, linalool treatment showed stronger promoting effects on PCD at the same time point, which may be caused by the higher ROS content inducing higher caspase-9-like and caspase-3-like activities in a short time.

Production of primary metabolites in Microcystis aeruginosa in regulation of nitrogen limitation.

The aim of this work was to study the regulatory effect of nitrogen (N) deficiency on primary metabolites in Microcystis aeruginosa, and promote the utilization of the alga. Low-N and Non-N conditions, especially Non-N, reduced the cell growth and photosynthetic abilities compared to Normal-N, as N deficiency triggered the down-regulation of genes involving in the photosynthetic process. Non-N not changed lipid content, due to no up-regulation of genes that promoted lipid synthesis. Soluble protein content significantly decreased under Non-N, which may result from the declined expression of genes relating to amino acid and histidyl-transfer RNA synthesis. Soluble and insoluble carbohydrate content significantly increased under Non-N, as the expression variation of genes blocked sugar degradation and promoted lipopolysaccharide synthesis. Therefore, M. aeruginosa can be used as the feedstock to produce carbohydrates under N deficiency for bioethanol production, and the remainder lipids after carbohydrate extraction can be used to produce biodiesel.

Algicidal properties of extracts from Cinnamomum camphora fresh leaves and their main compounds.

Plant allelochemicals are considered as the source of effective, economic and friendly-environmental algaecides. To uncover the anti-algal activities of Cinnamomum camphora fresh leaves and their main algicidal agents, we investigated the inhibitory effects of water and methanol extracts from C. camphora fresh leaves on Microcystis aeruginosa and Chlamydomonas reinhardtii cell growth, analyzed the composition of the water and methanol extracts, and determined the main compounds in extracts on the growth of the two algae and their anti-algal mechanism from photosynthetic abilities. Water and methanol extracts from C. camphora fresh leaves can inhibit M. aeruginosa and C. reinhardtii cell growth, and methanol extracts showed stronger inhibitory effects, due to their more compounds and higher molar concentration. There were 23 compounds in the water extracts, mainly including terpenoids, esters, alcohols, and ketones. Compared to the water extracts, 9 new compounds were detected in the methanol extracts, and the molar concentration of total compounds in methanol extracts increased by 1.3 folds. Camphor, α-terpineol and linalool were 3 main compounds in the water and methanol extracts. Their mixture (1: 3: 6) and individual compound showed remarkable inhibition on M. aeruginosa and C. reinhardtii cell growth. The degradation of photosynthetic pigments and the reduction of maximum quantum yield of photosystem II (PSII) photochemistry, coefficient of photochemical quenching as well as apparent electron transport rate in C. reinhardtii cells aggravated gradually with increasing the concentration of the mixture and individual compound, while the non-photochemical dissipation of absorbed light energy increased gradually, which led to the decline of photosynthetic abilities. This indicated that camphor, α-terpineol and linalool were 3 main algicidal agents in C. camphora fresh leaf extracts, and they inhibited algal growth by inducing photosynthetic pigment degradation and declining PSII efficiency. Therefore, C. camphora fresh leaf extracts and their main components have potential utilization values as algaecides.