HSFA3 functions as a positive regulator of HSFA2a to enhance thermotolerance in perennial ryegrass.

Perennial ryegrass (Lolium perenne) is a widely used cool season turfgrass with outstanding turf quality and grazing tolerance. High temperature is the key factor restricting the distribution of perennial ryegrass in temperate and sub-tropic regions. In this study, we found that one HEAT SHCOK TRANSCRIPTION FACOTR (HSF) class A gene from perennial ryegrass, LpHSFA3, was highly induced by heat stress. LpHSFA3 is localized in nucleus and functions as a transcription factor. Ectopic overexpression of LpHSFA3 in Arabidopsis improved thermotolerance and rescued heat sensitive deficiency of athsfa3 mutant. Overexpression of LpHSFA3 in perennial ryegrass enhanced heat tolerance and increased survival rate in summer season as evidenced by decreased EL and MDA, increased number of green leaves and total chlorophyll content. LpHSFA3 binds to the HSE region in LpHSFA2a promoter to constitutively activate the expression of LpHSFA2a and downstream heat stress responsive genes. Ectopic overexpression of LpHSFA2a consequently rescued thermal sensitivity of athsfa3 mutant and enhanced thermotolerance of athsfa2 mutant. Perennial ryegrass protoplasts with overexpression of LpHSFA3 and LpHSFA2a exhibited induction of similar subsets of heat responsive genes. These results indicated that transcription factor LpHSFA3 functions as positive regulator of LpHSFA2a to improve thermotolerance of perennial ryegrass, providing further evidence to understand the regulatory networks of plant heat stress response.

The R2R3-MYB Transcriptional Repressor TgMYB4 Negatively Regulates Anthocyanin Biosynthesis in Tulips (Tulipa gesneriana L.).

Anthocyanins play a paramount role in color variation and significantly contribute to the economic value of ornamental plants. The conserved activation complex MYB-bHLH-WD40 (MBW; MYB: v-myb avian myeloblastosis viral oncogene homolog; bHLH: basic helix-loop-helix protein; WD40:WD-repeat protein) involved in anthocyanin biosynthesis has been thoroughly researched, but there have been limited investigations into the function of repressor factors. In this study, we characterized TgMYB4, an R2R3-MYB transcriptional repressor which is highly expressed during petal coloration in red petal cultivars. TgMYB4-overexpressing tobaccos exhibited white or light pink petals with less anthocyanin accumulation compared to control plants. TgMYB4 was found to inhibit the transcription of ANTHOCYANIDIN SYNTHASE (TfANS1) and DIHYDRO-FLAVONOL-4-REDUCTASE (AtDFR), although it did not bind to their promoters. Moreover, the TgMYB4 protein was able to compete with the MYB activator to bind to the :bHLHprotein, thereby suppressing the function of the activator MBW complex. These findings demonstrate that TgMYB4 plays a suppressive role in the regulation of anthocyanin synthesis during flower pigmentation.

Transcription factors TgbHLH42-1 and TgbHLH42-2 positively regulate anthocyanin biosynthesis in Tulip (Tulipa gesneriana L.).

The floral coloration of tulip flowers is one of the most prominent traits contributing to its high ornamental value. The molecular mechanisms of petal coloration remain elusive in tulip species. In this study, we performed comparative metabolome and transcriptome analyses using four tulip cultivars with distinguished petal colors. Four types of anthocyanins were identified, including cyanidin derivatives and pelargonidin derivatives. Comparative transcriptome analysis identified 22,303 differential expressed genes (DEGs) from the four cultivars, and 2589 DEGs were commonly regulated in three comparison groups (colored vs. white cultivar), including anthocyanins biosynthesis-related genes and regulatory transcription factors. Two basic helix-loop-helix (bHLH) transcription factors, TgbHLH42-1 and TgbHLH42-2, with differential expression levels among cultivars and petal developmental stages, have high homology to TRANSPARENT TESTA 8 (AtTT8) of Arabidopsis. The anthocyanins accumulation in TgbHLH42-1 overexpressing (OE) seedlings was markedly greater than that in wild-type seedlings in the presence of methyl jasmonate (MeJA), but not for TgbHLH42-2 OE seedlings. Both TgbHLH42-1 and TgbHLH42-2 restored pigmentation defects in tt8 mutant seeds after complementation assay. TgbHLH42-1 could interact with MYB protein AtPAP1 to synergistically activate the transcription of AtDFR, whereas TgbHLH42-2 failed to. Silencing TgbHLH42-1 or TgbHLH42-2 individually could not, but simultaneously silencing both TgbHLH42 could reduce the anthocyanin in tulip petals. These results indicate that TgbHLH42-1 and TgbHLH42-2 function partially redundantly to positively regulate anthocyanin biosynthesis during tulip petal coloration.

De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli.

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization.

Genome-wide identification of heat shock transcription factor families in perennial ryegrass highlights the role of LpHSFC2b in heat stress response.

Perennial ryegrass (Lolium perenne) is a cool-season turf and forage grass. Heat shock transcription factors (HSFs) play an important role in regulating plant abiotic stress. However, HSFs in perennial ryegrass have rarely been characterized. Here, 25 LpHSFs were identified from the perennial ryegrass genome. Phylogenetic analysis showed that the LpHSFs could be classified into 12 subclasses. Gene structure analysis showed that 22 LpHSFs have only one intron. Cis-acting elements analysis revealed that the promoter of 15 LpHSFs contained hormone-responsive and abiotic stress-responsive elements. Expression profile analysis indicated that 24 LpHSFs were differentially expressed under submerge, drought, heat, and cold stresses. In addition, a subclass C2 gene, LpHSFC2b, was significantly induced by abiotic stresses. The LpHSFC2b protein is localized to the nucleus, and heterologous expression of LpHSFC2b in Arabidopsis improves plant thermotolerance. This study provides insights useful for the breeding of stress tolerance in perennial ryegrass.

Gene cloning, expression in E. coli, and in vitro refolding of a lipase from Proteus sp. NH 2-2 and its application for biodiesel production.

OBJECTIVE: To obtain active lipases for biodiesel production by refolding Proteus sp. lipase inclusion bodies expressed in E. coli. RESULTS: A lipase gene lipPN1 was cloned from Proteus sp. NH 2-2 and expressed in E. coli BL21(DE3). Non-reducing SDS-PAGE revealed that recombinant LipPN1(rLipPN1) were prone to form inclusion bodies as disulfide-linked dimers in E. coli. Site-directed mutagenesis confirmed that Cys85 in LipPN1 was involved in the dimer formation. After optimizing the inclusion body refolding conditions, the maximum lipase activity reached 1662 U/L. The refolded rLipPN1 exhibited highest activity toward p-nitrophenyl butyrate at pH 9.0 and 40 °C. It could be activated by Ca2+ with moderate tolerance to organic solvents. It could also convert soybean oil into biodiesel at a conversion ratio of 91.5%. CONCLUSION: Preventing the formation of disulfide bond could enhance the refolding efficiency of rLipPN1 inclusion bodies.

Pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth enhances superoxide anion-scavenging capacity of Cu/Zn-SOD.

A bioassay-guided fractionation of extract from Gluconobacter oxydans fermentation broth afforded Compound 1, which was identified as pyrroloquinoline quinone (PQQ) by spectroscopic methods. PQQ has been shown to enhance the superoxide anion-scavenging capacity significantly for Cu/Zn-SOD. To illustrate the mechanism, the interaction between PQQ and Cu/Zn-SOD was investigated. The multiple binding sites involving hydrogen bonds and van der Waals force between PQQ and Cu/Zn-SOD were revealed by isothermal titration calorimetry. The α-helix content was increased in the Cu/Zn-SOD structure with the addition of PQQ into the solution through ultraviolet (UV) spectroscopy. These results indicated that PQQ could change the conformation of Cu/Zn-SOD through interaction, which could enhance its superoxide anion-scavenging capacity. Therefore, PQQ is a potential natural antioxidant.

Characterization and phylogenetic analysis of the mating-type loci in the asexual ascomycete genus Ulocladium.

The genus Ulocladium is thought to be strictly asexual. Mating-type (MAT) loci regulate sexual reproduction in fungi and their study may help to explain the apparent lack of sexual reproduction in Ulocladium. We sequenced the full length of two MAT genes in 26 Ulocladium species and characterized the entire MAT idiomorphs plus flanking regions of Ulocladium botrytis. The MAT1-1 ORF encodes a protein with an alpha-box motif by the MAT1-1-1 gene and the MAT1-2 ORF encodes a protein with an HMG box motif by the MAT1-2-1 gene. Both MAT1-1-1 and MAT1-2-1 genes were detected in a single strain of every species. Moreover, the results of RT-PCR revealed that both MAT genes are expressed in all 26 Ulocladium species. This demonstrates that MAT genes of Ulocladium species might be functional and that they have the potential for sexual reproduction. Phylogenies based on MAT genes were compared with GAPDH and Alt a 1 phylograms in Ulocladium using maximum parsimony (MP) and Bayesian analysis. The MAT genealogies and the non-MAT trees displayed different topologies, indicating that MAT genes are unsuitable phylogenetic markers at the species level in Ulocladium. Furthermore, the conflicting topologies between MAT1-1-1 and MAT1-2-1 phylogeny indicate separate evolutionary events for the two MAT genes. However, the intergeneric phylogeny of four closely allied genera (Ulocladium, Alternaria, Cochliobolus, Stemphylium) based on MAT alignments demonstrated that MAT genes are suitable for phylogenetic analysis among allied genera.

GC-MS-based metabolomics study of the responses to arachidonic acid in Blakeslea trispora.

The potential use of arachidonic acid (AA) to enhance the production of ß-carotene in Blakeslea trispora was investigated in this work. To study the mechanism of the B. trispora response to AA, we used a systematic analytical approach to investigate the changes in the B. trispora cell metabolome at different time points after AA treatment. A maximum of ß-carotene production was obtained when 0.4g/l AA was added after 36h of cultivation. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach and a multivariate analysis were used to investigate the intracellular biochemical changes in B. trispora. With the aid of principal component analysis (PCA), the intracellular metabolite profiles of all the groups were distinguished. Moreover, a group classification and pairwise discrimination between the control and AA-treated groups were obtained through partial least-squares-discriminant analysis (PLS-DA), and 27 differential metabolites with variable importance in the projection (VIP) value higher than 1 were identified, which was also confirmed by the subsequent hierarchical cluster analysis (HCA). Separation of the control and AA-treated groups was mainly due to the compounds of the Krebs cycle, fatty acids and amino acids. With the treatment of AA, the glycolysis was enhanced and the use of glucose for fermentation was increased. The increased levels of some fatty acids and decreased levels of amino acids in the AA-treated cells could also be the responses to the addition of AA. Metabolomics provided a powerful methodology to gain insight in metabolic changes induced by metabolic stimulators in microorganisms.

Improved ß-carotene production by oxidative stress in Blakeslea trispora induced by liquid paraffin.

When 3 % (v/v) liquid paraffin was added to the medium, ß-carotene production increased from 397 to 715 mg l(-1) in mated cultures of Blakeslea trispora. Liquid paraffin also enhanced the oxygen concentration and induce high oxidative stress, as observed by the increase in activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). After 84 h of cultivation in the presence of liquid paraffin, the activities of SOD, CAT and POD in B. trispora increased 77, 52.5 and 76.6 %, respectively.

Improved ß-carotene biosynthesis and gene transcription in Blakeslea trispora with arachidonic acid.

With 0.4 g l(-1) arachidonic acid (AA) added to the medium after 36 h fermentation, ß-carotene production in mated cultures of Blakeslea trispora was 73 % higher than that of the control at 690 mg l(-1). With the treatment of AA, the transcriptional levels of genes hmgR, carRA and carB, that are involved in carotene biosynthesis, increased by 31, 22 and 39 %, respectively.