Yanling Bai

Acetic acid-induced programmed cell death and release of volatile organic compounds in Chlamydomonas reinhardtii

Acetic acid widely spreads in atmosphere, aquatic ecosystems containing residues and anoxic soil. It can inhibit aquatic plant germination and growth, and even cause programmed cell death (PCD) of yeast. In the present study, biochemical and physiological responses of the model unicellular green algae Chlamydomonas reinhardtii were examined after acetic acid stress. H(2)O(2) burst was found in C. reinhardtii after acetic acid stress at pH 5.0 for 10 min. The photosynthetic pigments were degraded, gross photosynthesis and respiration were disappeared gradually, and DNA fragmentation was also detected. Those results indicated that C. reinhardtii cells underwent a PCD but not a necrotic, accidental cell death event. It was noticed that C. reinhardtii cells in PCD released abundant volatile organic compounds (VOCs) upon acetic acid stress. Therefore, we analyzed the VOCs and tested their effects on other normal cells. The treatment of C. reinhardtii cultures with VOCs reduced the cell density and increased antioxidant enzyme activity. Therefore, a function of VOCs as infochemicals involved in cell-to-cell communication at the conditions of applied stress is suggested.

Overexpression of the Arabidopsis photorespiratory pathway gene, serine: glyoxylate aminotransferase ( AtAGT1 ), leads to salt stress tolerance in transgenic duckweed ( Lemna minor )

Salt stress has attracted increasing attention due to its toxic ability to restrict plant growth, and the photorespiration pathway has been shown to develop improved plant tolerance to abiotic stress. In this study, an Arabidopsis photorespiratory pathway gene serine: glyoxylate aminotransferase (SGAT), named as AtAGT1, was successfully overexpressed in duckweed (Lemna minor) to investigate the salinity defense capability in three transgenic overexpressed (OE) lines. Increased SGAT activity and decreased endogenous serine levels in these transgenic plant lines under salt stress resulted in enhanced protection against root abscission, higher maximum quantum yield of photosystem II (Fv/Fm), increased defense from cell damage as a result of improved cell membrane integrity, a decrease of reactive oxygen species (ROS) accumulation, and a strengthened antioxidant system. The salt tolerance in these transgenic OE lines indicates that the improvement of photorespiration stimulated the antioxidant system to scavenge ROS. The change of serine level also suggests the role of serine during salt stress. This transgenic engineering in duckweed not only introduced salt tolerance to this aquatic plant but also reveals a significant role of photorespiration during salinity stress.

Effects of NaCl and Na2CO3 stresses on photosynthetic ability of Chlamydomonas reinhardtii

Chloride and carbonate salts are the main salts causing salinization and widely exist in aquatic environment, so algae may suffer from salinization stress for high water evaporation. In this study, in order to investigate and compare the toxic effects of the two salts on algal photosynthesis, we used NaCl and Na2CO3 to stress Chlamydomonas reinhardtii. Under the two salt stresses, the content of O2−· and H2O2 in the cells was increased significantly, and it was much higher in Na2CO3 treatment than in NaCl treatment at the same Na+ concentration. The absorbance spectra and 4th derivative spectra of photosynthetic pigments were declined under 300 mM NaCl and 25 mM Na2CO3 stresses, and remarkably changed under 50 mM and 100 mM Na2CO3 stresses. When the cells stressed by the two salts, the maximum quantum yield (Fv/Fm), electron transport rate (ETR) and photochemical quenching (qP) were reduced markedly, but the nonphotochemical dissipation (NPQ) was increased markedly. At the same Na+ concentration, Na2CO3 stress had stronger toxic effects on photosynthetic ability than NaCl stress.

Reactive oxygen species contribute to the release of volatile organic compounds from Chlamydomonas reinhardtii during programmed cell death

Acetic acid at pH 5.0 can induce programmed cell death (PCD) in Chlamydomonas reinhardtii cells, and abundant volatile organic compounds (VOCs) were released during the process. In this study, the caspase‐3‐like activity was determined during the PCD, and it was increased significantly after 1 h. During the PCD, the dynamic release of VOCs from the cells was analyzed, and the emissions of total VOCs were raised markedly and reached the highest level at 2 h. Among the seven types of VOCs, such as alkanes, alkenes, terpenoids, alcohols, aldehydes, ketones and esters, three oxygenated compounds (aldehydes, ketones and esters) showed the most significant increase. O2‐· and H2O2 were rapidly accumulated to high levels in the cells at the beginning of the PCD, but their content was reduced during the process. The activities of antioxidant enzymes were reduced gradually and even disappeared completely, demonstrating that the reduction of reactive oxygen species (ROS) may not be scavenged by the antioxidant enzyme system. ROS have an intense oxidation and scavenging ability to volatile compounds, and the oxidation results in the production of oxygenated compounds. Therefore, the abundant production of oxygenated compounds indicated that ROS may play an important role in the dynamic release of VOCs from C. reinhardtii cells during PCD.

Study of amino acids as nitrogen source in Chlamydomonas reinhardtii

All five L‐amino acids tested (L‐serine, L‐lysine, L‐leucine, L‐cysteine and L‐arginine) were used by Chlamydomonas reinhardtii as sole nitrogen source. Among these, L‐Cys was special as it has not been reported before. While these amino acids could be used in the dark only in the presence of acetic acid, in conditions of light they could support the growth of C. reinhardtii without the supplementation of acetic acid. When cultured in the TAP‐N medium, the chlorophyll content was found to be lower in the dark, but higher in the light for the cells grown with L‐Arg than with other four amino acids. Exogenously supplied L‐Ser and L‐Lys did not accumulate in the cells, demonstrating that they were used by supplying ammonium to the cells from the activity of an extracellular deaminase. Further results showed that the induction of the extracellular deaminase activity required a period of nitrogen starvation, regardless of the medium containing acetic acid or not. Results also showed that the uptake of L‐Cys was similar to L‐Leu, most likely via passive diffusion. When L‐Cys and L‐Leu were supplied together to the nitrogen‐starved cells, the absorption of L‐Cys did not affect the uptake of L‐Leu.

Overexpression of PSP1 enhances growth of transgenic Arabidopsis plants under ambient air conditions

The importance of the phosphorylated pathway (PPSB) of l-serine (Ser) biosynthesis in plant growth and development has been demonstrated, but its specific role in leaves and interaction with photorespiration, the main leaf Ser biosynthetic pathway at daytime, are still unclear. To investigate whether changes in biosynthesis of Ser by the PPSB in leaves could have an impact on photorespiration and plant growth, we overexpressed PSP1, the last enzyme of this pathway, under control of the Cauliflower Mosaic Virus 35S promoter in Arabidopsis thaliana. Overexpressor plants grown in normal air displayed larger rosette diameter and leaf area as well as higher fresh and dry weight than the wild type. By contrast, no statistically significant differences to the wild type were observed when the overexpressor seedlings were transferred to elevated CO2, indicating a relationship between PSP1 overexpression and photorespiration. Additionally, the transgenic plants displayed higher photorespiration, an increase in CO2 net-uptake and stronger expression in the light of genes encoding enzymes involved in photorespiration. We further demonstrated that expression of many genes involved in nitrogen assimilation was also promoted in leaves of transgenic plants and that leaf nitrate reductase activity increased in the light, too, although not in the dark. Our results suggest a close correlation between the function of PPSB and photorespiration, and also nitrogen metabolism in leaves.

Chloroplast-Targeted Expression of Human sTRAIL Protein in Transgenic Tobacco

This study was aimed to express the soluble extracellular domain of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL), an important potential agent for cancer therapy, in transgenic tobacco. Transformation vectors were constructed either for targeting the protein into chloroplasts (35S:tp-strail) or for accumulating it in the cytosol (35S:strail). After Agrobacterium-mediated transformation of wild type tobacco with vector 35S:tp-strail or vector 35S:strail, 8 and 3 transgenic lines were obtained, respectively. The transcription of the recombinant gene encoding sTRAIL was analyzed by RT-PCR. And the expressed sTRAIL in transgenic tobacco plants was detected by both Western blot and ELISA analysis. The results showed that strail gene was transcribed in all transgenic lines for both untargeted and targeted expression. However, sTRAIL protein accumulation was only detected in transgenic lines transformed with vector 35S:tp-strail. Our preliminary results showed the sTRAIL expressed and accumulated in chloroplast of transgenic tobacco was biologically active. This is, to our knowledge, the first report of an active sTRAIL expressed in higher plant.

ROS production is associated with glycolate metabolism in Chlamydomonas reinhardtii (Chlorophyceae) under salt stress

Abstract: The photorespiration metabolism in unicellular algae is different from that in higher plants because of the absence of peroxisomes, and conversion of glycolate to glyoxylate is catalyzed by glycolate dehydrogenase without the production of reactive oxygen species (ROS). It has been reported that the glycolate–quinone oxidoreductase system (GQOS) also plays a role in the formation of glyoxylate from glycolate that is coupled to photosynthetic electron transport. In this study, we showed that production of ROS was associated with the oxidation of glycolate in chloroplasts of Chlamydomonas reinhardtii cells and that both ROS accumulation and photorespiration were promoted by NaCl stress. Salicylhydroxamic acid (SHAM) and methoxylamine (MOA) are inhibitors that block effectively the GQOS oxidation of glycolate and glycine decarboxylase complex, respectively. Our data showed that in the dark, whether or not the cells were treated with inhibitor, no remarkable difference was found in ROS levels under the same NaCl stress. In the light, however, the ROS level in the cells treated with SHAM or MOA was significantly lower than that in nontreated cells, with SHAM being more effective. The increase in serine:glyoxylate aminotransferase (SGAT) activity and glycine/serine (Gly/Ser) ratio were 8.3% and 30.8% under 50 mM, and reached 34.4% and 1.5-fold under 100 mM NaCl stress.