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.

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.

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.

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.

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.

Inhibitory effects of extracts from Cinnamomum camphora fallen leaves on algae.

Natural allelochemicals are considered as a source of algaecides. To uncover the anti-algal activity of Cinnamomum camphora fallen leaves and promote their usage as algaecides, the composition of their water and methanol extracts was analyzed, and the inhibitory effects of extracts on the growth of Microcystis aeruginosa and Chlamydomonas reinhardtii, and chlorophyll (Chl) content and photosynthetic abilities in C. reinhardtii were investigated. Twenty-five compounds were detected in the water extracts, mainly including terpenoids, esters, alcohols, and ketones. Compared to water extracts, there were more compounds and higher concentration in methanol extracts. Both water and methanol extracts inhibited the growth of the two algae, and 15 mg·ml-1 methanol extracts killed the algal cells after 48 h. The levels of Chl a and Chl b, as well as maximum quantum yield of photosystem II photochemistry (Fv/Fm) in C. reinhardtii cells reduced gradually with increasing the concentration of extracts, while the maximum quantum yield of non-photochemical de-excitation (φDO) increased gradually. At the same concentration, methanol extracts showed stronger inhibitory effects than water extracts, due to their higher number of compounds and higher concentration. Therefore, C. camphora fallen leaves have a potential value as an algaecide.