Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.     

Comparative proteomics and physiological characterization of Arabidopsis thaliana seedlings in responses to Ochratoxin A

Ochratoxin A (OTA) is a mycotoxin that is primarily produced by Aspergillus ochraceus and Penicillium verrucosum. This mycotoxin is a contaminant of food and feedstock worldwide and may induce cell death in plants. To investigate the dynamic growth process of Arabidopsis seedlings in response to OTA stress and to obtain a better understanding of the mechanism of OTA toxicity towards Arabidopsis, a comparative proteomics study using 2-DE and MALDI-TOF/TOF MS/MS was performed. Mass spectrometry analysis identified 59 and 51 differentially expressed proteins in seedlings exposed to 25 and 45 μM OTA for 7 days, respectively. OTA treatment decreased root elongation and leaf area, increased anthocyanin accumulation, damaged the photosynthetic apparatus and inhibited photosynthesis. Treatment of the seedlings with 25 μM OTA enhanced energy metabolism, whereas higher concentration of OTA (45 μM) inhibited energy metabolism in the seedlings. OTA treatment caused an increase of ROS, an enhancement of antioxidant enzyme defense responses, disturbance of redox homeostasis and activation of lipid oxidation. Glutamine and S-adenosylmethionine metabolism may also play important roles in the response to OTA. In conclusion, our study provided novel insights regarding the response of Arabidopsis to OTA at the level of the proteome. These results are expected to be highly useful for understanding the physiological responses and dissecting the OTA response pathways in higher plants.     

Antrodia camphorata ATCC 200183 sporulates asexually in submerged culture

Antrodia camphorata is a well-known Chinese medicinal mushroom that protects against diverse health-related conditions. Submerged fermentation of A. camphorata is an alternative choice for the effective production of bioactive metabolites, but the effects of nutrition and environment on mycelial morphology are largely unknown. In this study, we show that A. camphorata American Type Culture Collection 200183 can form arthrospores in the end of liquid fermentation. Different morphologies of A. camphorata in submerged culture were analyzed using scanning electron microscopy. The optimal carbon and nitrogen sources for sporulation were soluble starch and yeast extract. We found that a carbon-to-nitrogen ratio (C/N) of 40:1, MgSO₄ (0.5 g/l), KH₂PO₄ (3.0 g/l), an initial pH?5.0, and an inoculum size of 1.5?×?10⁵ spores/ml led to maximum production of arthroconidia. Our results will be useful in the regulation and optimization of A. camphorata cultures for efficient production of arthroconidia in submerged culture, which can be used as inocula in subsequent fermentation processes.     

Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium

High acetate accumulation was produced during glucose fermentation in high cell density cultures, which is harmful to cell growth. In order to reduce the negative impact of acetate accumulation on the fermentation products, we introduced the Escherichia coli acetyl-CoA synthetase (ACS) gene into the marine microalga Schizochytrium sp. TIO1101, generating genetically modified ACS transformants. The results of PCR and blotting analyses showed that the exogenous ACS gene was incorporated into the genome and successfully expressed. The engineered Schizochytrium increased the pH value and reduced the acetate concentration in the final fermentation medium significantly. Furthermore, the ACS transformants exhibited faster growth and glucose consumption rates than the wild-type strain. The biomass and fatty acid proportion of ACS transformants increased by 29.9 and 11.3 %, respectively. Taken together, the data suggest that ACS overexpression in Schizochytrium might improve the utilization of carbon resource and decrease the production of acetate byproduct. These results demonstrate that application of ACS in metabolic genetic engineering could improve the properties of Schizochytrium significantly.     

In situ hydrogen utilization for high fraction acetate production in mixed culture hollow-fiber membrane biofilm reactor

Syngas fermentation is a promising route for resource recovery. Acetate is an important industrial chemical product and also an attractive precursor for liquid biofuels production. This study demonstrated high fraction acetate production from syngas (H₂ and CO₂) in a hollow-fiber membrane biofilm reactor, in which the hydrogen utilizing efficiency reached 100 % during the operational period. The maximum concentration of acetate in batch mode was 12.5 g/L, while the acetate concentration in continuous mode with a hydraulic retention time of 9 days was 3.6?±?0.1 g/L. Since butyrate concentration was rather low and below 0.1 g/L, the acetate fraction was higher than 99 % in both batch and continuous modes. Microbial community analysis showed that the biofilm was dominated by Clostridium spp., such as Clostridium ljungdahlii and Clostridium drakei, the percentage of which was 70.5 %. This study demonstrates a potential technology for the in situ utilization of syngas and valuable chemical production.     

Characterization of an ammonium transporter in the oleaginous alga Chlorella protothecoides

A suppression subtractive hybridization cDNA library was used to screen the differently expressed (up-regulated) genes in the photosynthesis–fermentation approach (PFA) of Chlorella protothecoides cultivation. A total of 87 clones were obtained and sequenced, in which 78 clones were homologous to known genes in databases. Among them, the ammonium transporter gene (CpAMT1) was characterized in detail. Quantitative real-time PCR showed that the expression of CpAMT1 was significantly induced by PFA and correlated with lipid accumulation. The up-regulation of CpAMT1 was suppressed by glutamine, while the lipid biosynthesis was also inhibited. Further analysis showed that the expression of CpAMT1 was correlated with glutamine synthetase activity, suggesting that CpAMT1, along with glutamine synthetase/glutamate synthase, may be responsible for nitrogen sensing in C. protothecoides. Together, these results imply that the ammonium transporter CpAMT1 could be the initial sensor of nitrogen deficiency and channels the carbon excess toward lipid biosynthesis.     

Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH?6.5 and 65 °C, RmXEG12B at pH?5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U?mg^?1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-d-Glc residue, X is a xylosylated β-d-Glc residue, and L is a β-d-Glc residue substituted by xylosyl-galactose).