Limei Chen

Assimilation of Formaldehyde in Transgenic Plants Due to the Introduction of the Bacterial Ribulose Monophosphate Pathway Genes

Formaldehyde (HCHO) is an air pollutant suspected of being carcinogenic and a cause of sick-house syndrome. Microorganisms called methylotrophs, which can utilize reduced C1 compounds such as methane and methanol, fix and assimilate HCHO, whereas most plants are unable to assimilate HCHO directly. We found that a bacterial formaldehyde-fixing pathway (ribulose monophosphate pathway) can be integrated as a bypass to the Calvin-Benson cycle in transgenic Arabidopsis thaliana and tobacco by genetic engineering. These plants showed enhanced tolerance to HCHO and enhanced capacity to eliminate gaseous HCHO by fixing it as a sugar phosphate. Our results provide a novel strategy for phytoremediation of HCHO pollution, and also represent the first step toward the production of plants that can assimilate natural gas-derived C1 compounds.

Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L)

Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein.

Overexpression of Arabidopsis Dof1, GS1 and GS2 Enhanced Nitrogen Assimilation in Transgenic Tobacco Grown Under Low-Nitrogen Conditions

Nitrogen metabolism is essential for plant growth and development. The glutamine synthetase (GS) enzyme and the Dof transcription factor play important roles in the regulation of nitrogen metabolism. Using expression vectors, transgenic tobacco lines overexpressing each of Dof1 (Dof1.7), GS1 (GLN1;4) and GS2 were developed. cDNA clones of each of these transgenes were derived from Arabidopsis thaliana, and each transgene was under the control of the Rubisco small subunit promoter, PrbcS—a leaf-specific light-inducible promoter. Under low-nitrogen conditions, plant length, leaf surface, total protein, chlorophyll, as well as sucrose and glucose contents in leaves of transgenic tobacco lines were higher than those of wild-type plants. In addition, transgenic plants exhibited increased activities of enzymes involved in carbon and nitrogen metabolism, such as GS, nitrate reductase (NR), phosphoenolpyruvate carboxylase (PEPC) and pyruvate kinase (PK). The contents of most amino acids were significantly higher in transgenic plants than those of wild-type plants, whereas levels of nitrate, malic acid, and citric acid were significantly lower than those of wild-type plants. These findings suggest that Dof1 and GS work together to regulate the nitrogen metabolic pathway in plants, and this enhances nitrogen assimilation in transgenic tobacco plants grown under low-nitrogen conditions.

Transcriptional and physiological changes of alfalfa in response to aluminium stress

Medicago sativa is an excellent pasture legume, but it is very sensitive to aluminium (Al) toxicity. To better understand the mechanism of M. sativa sensitivity to Al, a forward suppression subtractive hybridization (SSH) cDNA library for an Al-sensitive cultivar, M. sativa L. cv. Yumu No. 1 (YM1), under 5 μ m Al stress over a 24 h period was constructed to analyse changes in its gene expression in response to Al stress. Sequence analysis for the SSH cDNA library generated 291 high-quantity expression sequence tags (ESTs). Of these, 229 were known as functional ESTs, 137 of which have already been reported as Al response genes, whereas the other 92 were potentially novel Al-associated genes. The up-regulation of known Al resistance-associated genes encoding the transcription factor sensitive to proton rhizotoxicity 1 ( STOP1 ) and malate transporter MsALMT1 (Al-activated malate transporter) as well as genes for antioxidant enzymes was observed. Reverse transcription polymerase chain reaction analysis validated the reliability of the SSH data and confirmed the up-regulated expression of STOP1 and MsALMT1 under 5 μ m Al stress. The analysis of physiological changes indicated that hydrogen peroxide (H 2 O 2 ) and malondialdehyde levels were elevated rapidly under 5 μ m Al stress, suggesting that severe oxidative stress occurred in the YM1 roots. The up-regulation of antioxidant-related genes might be an important protective mechanism for YM1 in response to the oxidative stress induced by 5 μ m Al toxicity. Al-induced malate exudation was increased drastically during the early period after Al treatment, which might have been due to the up-regulation and function of MsALMT and STOP1 . However, malate exudation from the YM1 roots declined quickly during the subsequent period, and a gradual decrease in malate content was simultaneously observed in the YM1 roots. This result is in agreement with the observation that organic acid metabolism-associated enzymes such as phosphoenolpyruvate carboxylase, citrate synthase and malate dehydrogenase were not present in the SSH library. This might be a major reason for the YM1 sensitivity to Al.

Overexpression of an HPS/PHI fusion enzyme from Mycobacterium gastri in chloroplasts of geranium enhances its ability to assimilate and phytoremediate formaldehyde.

Abstract3-Hexulose-6-phosphate synthase (HPS) and 6-phosphate-3-hexuloisomerase (PHI) are two key enzymes in the formaldehyde (HCHO) assimilation pathway in methylotrophs. The HPS/PHI fusion protein, encoded by the chimeric gene of hps and phi from Mycobacterium gastri MB19, possesses both HPS and PHI activities in an Escherichia coli transformant. Overexpression of the fusion protein in chloroplasts of geranium (Pelargonium sp. Frensham) created a photosynthetic HCHO assimilation pathway according to 13C-NMR analysis. The transgenic plants exhibited an enhanced ability in HCHO-uptake and [14C]HCHO-assimilation. Moreover, the transgenic plants showed greater HCHO-resistance and stronger capacity in purification of the HCHO-polluted air. Therefore, the use of the single chimeric gene may not only greatly simplify the transformation procedure but also improve the efficiency of phytoremediating HCHO in ornamental plants.

Changes in the Activity and Transcription of Antioxidant Enzymes in Response to Al Stress in Black Soybeans

In this study, the effects of Al stress on the activity and transcription of antioxidant enzymes were investigated in an acid-resistant black soybean (RB) and an acid-sensitive black soybean (SB) under hydroponic conditions to further clarify the role of antioxidant enzymes in the plant’s response to Al stress. The results indicated that oxidative stress was induced in the roots and leaves of RB and SB and that the stress level was higher in SB than in RB. Changes in the catalase (CAT) activity in response to Al stress occurred faster in RB roots and leaves than in SB. As the duration of Al stress increased, the peroxidase (POD) activity was enhanced more pronouncedly in RB roots and leaves than in SB. The activity of superoxide dismutase (SOD) in the roots and leaves of RB and SB was not responsive to Al stress. A high transcription level of a selected POD gene was detected in RB leaves, but no transcription of this POD gene was observed in SB leaves under Al stress. Moreover, the transcription level of this POD gene was higher in RB roots than in SB roots. Under Al stress, the transcription of two selected SOD genes showed an increasing trend in RB but decreased in SB. Furthermore, the transcription levels of these two selected SOD genes were always higher in RB than in SB. The above results suggest that not only does RB have a higher level of antioxidant enzyme activities but also that antioxidant enzyme genes can be upregulated by Al stress. This may be an important mechanism for RB to deal with oxidative stress induced by Al toxicity.

C1 metabolism and the Calvin cycle function simultaneously and independently during HCHO metabolism and detoxification in Arabidopsis thaliana treated with HCHO solutions

Formaldehyde (HCHO) is suggested to be detoxified through one-carbon (C1) metabolism or assimilated by the Calvin cycle in plants. To further understand the function of the Calvin cycle and C1 metabolism in HCHO metabolism in plants, HCHO elimination and metabolism by Arabidopsis thaliana in HCHO solutions was investigated in this study. Results verified that Arabidopsis could completely eliminate aqueous HCHO from the HCHO solutions. Carbon-13 nuclear magnetic resonance ((13)C-NMR) analysis showed that H(13)CHO absorbed by Arabidopsis was first oxidized to H(13)COOH. Subsequently, a clear increase in [U-(13)C]Gluc peaks accompanied by a strong enhancement in peaks of [2-(13)C]Ser and [3-(13)C]Ser appeared in Arabidopsis. Pretreatment with cyclosporin A or L-carnitine, which might inhibit the transport of (13)C-enriched compounds into chloroplasts and mitochondria, caused a remarkable decline in yields of both [U-(13)C]Gluc and [3-(13)C]Ser in H(13)CHO-treated Arabidopsis. These results suggested that both the Calvin cycle and the C1 metabolism functioned simultaneously during HCHO detoxification. Moreover, both functioned more quickly under high H(13)CHO stress than low H(13)CHO stress. When a photorespiration mutant was treated in 6 mm H(13)CHO solution, formation of [U-(13)C]Gluc and [2-(13)C]Ser was completely inhibited, but generation of [3-(13)C]Ser was not significantly affected. This evidence suggested that the Calvin cycle and C1 metabolism functioned independently in Arabidopsis during HCHO metabolism.

Physiological and molecular responses of broad bean (Vicia faba L.) to aluminum stress

In this study, the responses of broad bean cultivars resistant (YD) and sensitive (AD) to aluminum (Al) stress were investigated at physiological and molecular levels. The results showed that Al induced more citrate exudation in YD roots than that in AD roots, suggesting that citrate exudation is involved in broad bean Al resistance. The analyses for oxidative stress levels and antioxidant enzyme activities indicated that YD had a strong ability to cope with the oxidative stress induced by Al. To investigate the molecular responses of broad bean to Al stress further, a forward suppression subtractive hybridization cDNA library was constructed to identify Al-responsive genes in YD roots treated with 50-μM Al for a 24-h period. Of the obtained 162 high-quality ESTs, genes related to antioxidant enzymes including copper-zinc superoxide dismutase (SOD), class III peroxidase (POD) and germin-like protein (GEP) were up-regulated. Higher transcription levels of SOD and POD were observed in YD but not in AD roots, which is in agreement with the enhanced activities of antioxidant enzymes in YD roots under Al stress conditions. Furthermore, the up-regulated expression of vha2, encoding a plasma membrane (PM) H+-ATPase, and 14-3-3b in YD roots under Al stress were also detected and confirmed by RT-PCR analysis. Western and immunoprecipitation analyses indicated that Al-enhanced expressions and interactions of the PM H+-ATPase and 14-3-3 proteins might be involved in the regulation of citrate secretion in YD roots under Al stress.

Overexpression of spinach non-symbiotic hemoglobin in Arabidopsis resulted in decreased NO content and lowered nitrate and other abiotic stresses tolerance.

A class 1 non-symbiotic hemoglobin family gene, SoHb, was isolated from spinach. qRT-PCR showed that SoHb was induced by excess nitrate, polyethylene glycol, NaCl, H2O2, and salicylic acid. Besides, SoHb was strongly induced by application of nitric oxide (NO) donor, while was suppressed by NO scavenger, nitrate reductase inhibitor, and nitric oxide synthase inhibitor. Overexpression of SoHb in Arabidopsis resulted in decreased NO level and sensitivity to nitrate stress, as shown by reduced root length, fresh weight, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm), and higher malondialdehyde contents. The activities and gene transcription of superoxide dioxidase, and catalase decreased under nitrate stress. Expression levels of RD22, RD29A, DREB2A, and P5CS1 decreased after nitrate treatment in SoHb-overexpressing plants, while increased in the WT plants. Moreover, SoHb-overexpressing plants showed decreased tolerance to NaCl and osmotic stress. In addition, the SoHb-overexpression lines showed earlier flower by regulating the expression of SOC, GI and FLC genes. Our results indicated that the decreasing NO content in Arabidopsis by overexpressing SoHb might be responsible for lowered tolerance to nitrate and other abiotic stresses.

Purification Capability of Tobacco Transformed with Enzymes from a Methylotrophic BACTERIUM for Formaldehyde

Plants have the ability to remediate environmental pollution. Especially, they have a high purification capability for airpollution. We have measured the purification characteristics of foliage plants for indoor airpollutants—for example, formaldehyde (HCHO), toluene, and xylene—using a tin oxide gas sensor. HCHO is an important intermediate for biological fixation of C1 compounds in methylotrophs. The ribulose monophosphate pathway of HCHO fixation is inherent in many methylotrophic bacteria, which can grow on C1 compounds. Two genes for the key enzymes, HPS and PHI, from the methylotrophic bacterium Mycobacterium gastri MB19 were introduced into tobacco. In this article, the HCHO-removal characteristic of the transformant was examined by using the gas sensor in order to evaluate quantitatively. The purification characteristics of the transformant for toluene, xylene, and styrene were also measured. The results confirmed an increase of 20% in the HCHO-removal capability. The differences of the purification capabilities for toluene, xylene, and styrene were not recognized.