Transcription factors BZR1 and PAP1 cooperate to promote anthocyanin biosynthesis in Arabidopsis shoots.

Anthocyanins play critical roles in protecting plant tissues against diverse stresses. The complicated regulatory networks induced by various environmental factors modulate the homeostatic level of anthocyanins. Here, we show that anthocyanin accumulation is induced by brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana) shoots and shed light on the underlying regulatory mechanism. We observed that anthocyanin levels are altered considerably in BR-related mutants, and BRs induce anthocyanin accumulation by up-regulating the expression of anthocyanin biosynthetic genes. Our genetic analysis indicated that BRASSINAZOLE RESISTANT 1 (BZR1) and PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) are essential for BR-induced anthocyanin accumulation. The BR-responsive transcription factor BZR1 directly binds to the PAP1 promoter, regulating its expression. In addition, we found that intense anthocyanin accumulation caused by the pap1-D dominant mutation is significantly reduced in BR mutants, implying that BR activity is required for PAP1 function after PAP1 transcription. Moreover, we demonstrated that BZR1 physically interacts with PAP1 to cooperatively regulate the expression of PAP1 target genes, such as TRANSPARENT TESTA 8 (TT8), DIHYDROFLAVONOL 4-REDUCTASE (DFR), and LEUKOANTHOCYANIDIN DIOXYGENASE (LDOX). Our findings indicate that BZR1 functions as an integral component of the PAP1-containing transcription factor complex, contributing to increased anthocyanin biosynthesis. Notably, we also show that functional interaction of BZR1 with PAP1 is required for anthocyanin accumulation induced by low nitrogen stress. Taken together, our results demonstrate that BR-regulated BZR1 promotes anthocyanin biosynthesis through cooperative interaction with PAP1 of the MBW complex.

Comparative analysis of BZR1/BES1 family transcription factors in Arabidopsis.

Brassinazole Resistant 1 (BZR1) and bri1 EMS Suppressor 1 (BES1) are key transcription factors that mediate brassinosteroid (BR)-responsive gene expression in Arabidopsis. The BZR1/BES1 family is composed of BZR1, BES1, and four BES1/BZR1 homologs (BEH1-BEH4). However, little is known about whether BEHs are regulated by BR signaling in the same way as BZR1 and BES1. We comparatively analyzed the functional characteristics of six BZR1/BES1 family members and their regulatory mechanisms in BR signaling using genetic and biochemical analyses. We also compared their subcellular localizations regulated by the phosphorylation status, interaction with GSK3-like kinases, and heterodimeric combination. We found that all BZR1/BES1 family members restored the phenotypic defects of bri1-5 by their overexpression. Unexpectedly, BEH2-overexpressing plants showed the most distinct phenotype with enhanced BR responses. RNA-Seq analysis indicated that overexpression of both BZR1 and BEH2 regulates BR-responsive gene expression, but BEH2 has a much greater proportion of BR-independent gene expression than BZR1. Unlike BZR1 and BES1, the BR-regulated subcellular translocation of the four BEHs was not tightly correlated with their phosphorylation status. Notably, BEH1 and BEH2 are predominantly localized in the nucleus, which induces the nuclear accumulation of other BZR1/BES1 family proteins through heterodimerization. Altogether, our comparative analyses suggest that BEH1 and BEH2 play an important role in the functional interaction between BZR1/BES1 family transcription factors.

Inhibition of 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE expression by brassinosteroid reduces carotenoid accumulation in Arabidopsis.

Unlike the indispensable function of the steroid hormone brassinosteroid (BR) in regulating plant growth and development, the metabolism of secondary metabolites regulated by BR is not well known. Here we show that BR reduces carotenoid accumulation in Arabidopsis seedlings. BR-deficient or BR-insensitive mutants accumulated higher content of carotenoids than wild-type plants, whereas BR treatment reduced carotenoid content. We demonstrated that BR transcriptionally suppresses 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) expression involved in carotenogenesis via plastoquinone production. We found that the expression of HPPD displays an oscillation pattern that is expressed more strongly in dark than in light conditions. Moreover, BR appeared to inhibit HPPD expression more strongly in darkness than in light, leading to suppression of a diurnal oscillation of HPPD expression. BR-responsive transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) directly bound to the promoter of HPPD, and HPPD suppression by BR was increased in the bzr1-1D gain-of-function mutation. Interestingly, dark-induced HPPD expression did not cause carotenoid accumulation, due to down-regulation of other carotenoid biosynthetic genes in the dark. Our results suggest that BR regulates different physiological responses in dark and light through inhibition of HPPD expression.

Plant U-Box40 Mediates Degradation of the Brassinosteroid-Responsive Transcription Factor BZR1 in Arabidopsis Roots.

Brassinosteroid (BR) regulates a wide range of physiological responses through the activation of BRASSINAZOLE RESISTANT1 (BZR1), whose activity is tightly controlled by its phosphorylation status and degradation. Although BZR1 appears to be degraded in distinct ways in response to different hormonal or environmental cues, little is known about how BR signaling regulates its degradation. Here we show that the BR-regulated U-box protein PUB40 mediates the proteasomal degradation of BZR1 in a root-specific manner in Arabidopsis (Arabidopsis thaliana). BZR1 levels were strongly reduced by plant U-box40 (PUB40) overexpression, whereas the pub39 pub40 pub41 mutant accumulated much more BZR1 than wild type in roots. The bzr1-1D gain-of-function mutation reduced the interaction with PUB40, which suppressed PUB40-mediated BZR1 degradation in roots. The cell layer-specific expression of PUB40 in roots helps induce selective BZR1 accumulation in the epidermal layer. Both BR treatment and loss-of-function of PUB40 expanded BZR1 accumulation to most cell layers. In addition, BZR1 accumulation increased the resistance of pub39 pub40 pub41 to low inorganic phosphate availability, as observed in bzr1-1D BRASSINOSTEROID-INSENSITIVE2-induced phosphorylation of PUB40, which mainly occurs in roots, gives rise to BZR1 degradation through enhanced binding of PUB40 to BZR1 and PUB40's stability. Our results suggest a molecular mechanism of root-specific BZR1 degradation regulated by BR signaling.

Engineering of Corynebacterium glutamicum for Consolidated Conversion of Hemicellulosic Biomass into Xylonic Acid.

Xylonic acid is a promising platform chemical with various applications in the fields of food, pharmaceuticals, and agriculture. However, in the current process, xylonic acid is mainly produced by the conversion of xylose, whose preparation requires substantial cost and time. Here, Corynebacterium glutamicum is engineered for the consolidated bioconversion of hemicellulosic biomass (xylan) into xylonic acid in a single cultivation. First, for the efficient conversion of xylose to xylonic acid, xylose dehydrogenase (Xdh) and xylonolactonase (XylC) from Caulobacter crescentus are evaluated together with a previously optimized xylose transporter module (XylE of Escherichia coli), and cells expressing xdh and xylE genes with an optimized expression system can produce xylonic acid from xylose with 100% conversion yield. Next, to directly process xylan as a substrate, an engineered xylan-degrading module is introduced, in which two xylan-degrading enzymes (endoxylanase and xylosidase) are secreted into the culture medium. The engineered C. glutamicum successfully produce 6.23 g L-1 of xylonic acid from 20 g L-1 of xylan. This is the first report on xylonic acid production in C. glutamicum and this robust system will contribute to development of an industrially relevant platform for production of xylonic acid from raw biomass.

High-performance shape-engineerable thermoelectric painting.

Output power of thermoelectric generators depends on device engineering minimizing heat loss as well as inherent material properties. However, the device engineering has been largely neglected due to the limited flat or angular shape of devices. Considering that the surface of most heat sources where these planar devices are attached is curved, a considerable amount of heat loss is inevitable. To address this issue, here, we present the shape-engineerable thermoelectric painting, geometrically compatible to surfaces of any shape. We prepared Bi2Te3-based inorganic paints using the molecular Sb2Te3 chalcogenidometalate as a sintering aid for thermoelectric particles, with ZT values of 0.67 for n-type and 1.21 for p-type painted materials that compete the bulk values. Devices directly brush-painted onto curved surfaces produced the high output power of 4.0 mW cm-2. This approach paves the way to designing materials and devices that can be easily transferred to other applications.

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)-dependent secretion pathway by deletion of rrsE in Escherichia coli.

Although signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion, its limited capacity for the protein secretion is a major hurdle for utilizing the pathway as an attractive route for secretory production of recombinant proteins. Therefore, we developed an Escherichia coli mutant, whose efficiency of secretion via the SRP pathway was dramatically increased. First, we developed a novel FACS-based screening system by combining a periplasmic display system (PECS) and direct fluorescent labeling with the organoarsenic compound, FlAsH-EDT2 . With this screening system, transposon-insertion library of E. coli was screened, and then we isolated mutants which exhibited higher protein production through the SRP pathway than the parental strain. From the genetic analysis, we found that all isolated mutants had the same mutation-disruption of the 16S rRNA gene (rrsE). The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP-dependent proteins, antibodies, and G protein-coupled receptor. For the large-scale production of IgG and GPCR, we performed fed-batch cultivation with the rrsE-deficient mutant, and very high yields of IgG (0.4 g/L) and GPCR (1.4 g/L) were obtained. Biotechnol. Bioeng. 2016;113: 2453-2461. © 2016 Wiley Periodicals, Inc.

Modular Optimization of a Hemicellulose-Utilizing Pathway in Corynebacterium glutamicum for Consolidated Bioprocessing of Hemicellulosic Biomass.

Hemicellulose, which is the second most abundant polysaccharide in nature after cellulose, has the potential to become a major feedstock for microbial fermentation to produce various biofuels and chemicals. To utilize hemicellulose economically, it is necessary to develop a consolidated bioprocess (CBP), in which all processes from biomass degradation to the production of target products occur in a single bioreactor. Here, we report a modularly engineered Corynebacterium glutamicum strain suitable for CBP using hemicellulosic biomass (xylan) as a feedstock. The hemicellulose-utilizing pathway was divided into three distinct modules, and each module was separately optimized. In the module for xylose utilization, the expression level of the xylose isomerase (xylA) and xylulokinase (xylB) genes was optimized with synthetic promoters of different strengths. Then, the module for xylose transport was engineered with combinatorial sets of synthetic promoters and heterologous transporters to achieve the fastest cell growth rate on xylose (0.372 h(-1)). Next, the module for the enzymatic degradation of xylan to xylose was also engineered with different combinations of promoters and signal peptides to efficiently secrete both endoxylanase and xylosidase into the extracellular medium. Finally, each optimized module was integrated into a single plasmid to construct a highly efficient xylan-utilizing pathway. Subsequently, the direct production of lysine from xylan was successfully demonstrated with the engineered pathway. To the best of our knowledge, this is the first report of the development of a consolidated bioprocessing C. glutamicum strain for hemicellulosic biomass.

Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum.

Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum.

Effects of cranial electrotherapy stimulation on preoperative anxiety, pain and endocrine response.

OBJECTIVES: Cranial electrotherapy stimulation (CES) is used as a treatment for depression and anxiety, and as an adjunctive intervention for pain management. This prospective study investigated whether CES could decrease preoperative anxiety, the injection pain of rocuronium, postoperative pain and stress hormone levels. METHODS: Female patients undergoing thyroidectomy were randomly assigned to two groups, to receive either no pretreatment (control group) or CES pretreatment. Anxiety score, withdrawal response on rocuronium injection, and pain scores at 1, 4, 12 and 24 h post surgery were evaluated. Adrenocorticotrophic hormone (ACTH), cortisol and glucose levels were measured. Patients were blinded to the treatment condition. RESULTS: Fifty patients entered the study (n = 25 per group). Anxiety score and withdrawal responses during rocuronium injection were significantly reduced in the CES group compared with the control group. Pain score was significantly lower in the CES group than in the control group, 1 h and 4 h post surgery. There were no significant differences in ACTH, cortisol and glucose levels. CONCLUSIONS: CES pretreatment appears to reduce the level of preoperative anxiety, injection pain of rocuronium and postoperative pain. However, CES pretreatment did not affect stress hormone responses.

A case of malignant hyperthermia during anesthesia induction with sevoflurane -A case report-.

We experienced a case of malignant hyperthermia (MH) in 6-year-old boy during anesthesia induction for strabismus surgery. It has been generally reported that sevoflurane can induce the delayed onset of MH in the absence of succinylcholine. Our case of MH was elicited after about 2-3 min of sevoflurane administration with N(2)O, O(2) and rocuronium. However, we successfully treated the patient by early recognition of his condition and administering symptomatic treatment and dantrolene.

Factors determining early left atrial reverse remodeling after mitral valve surgery.

This study aimed to investigate the factors determining early left atrial (LA) reverse remodeling after mitral valve (MV) surgery. The left atrium is frequently dilated in patients with mitral stenosis (MS) or mitral regurgitation (MR). MV surgery usually results in LA volume reduction. However, the factors associated with LA reverse remodeling after MV surgery are not clearly defined. One hundred thirty-eight patients (51 men, 87 women; mean age, 53 years) underwent transthoracic echocardiography before and after MV surgery. Maximal LA volume was measured using the prolate ellipsoid model. The percentage of LA volume change was calculated. The patients were grouped according to age (<50 vs >or=50 years), predominant lesion (pure MR vs some degree of MS), type of surgery (MV repair vs MV replacement), and preoperative rhythm (sinus rhythm vs atrial fibrillation). LA volume decreased from 147+/-93 to 103+/-43 ml (p<0.001) after surgery. LA reverse remodeling was more prominent in patients who were <50 years old (percentage of LA volume change -31.2+/-17.4 vs -18.4+/-19.2, p<0.001), had pure MR (percentage of LA volume change -30.4+/-18.6 vs -17.3+/-18.2, p<0.001), and had a preoperative sinus rhythm (percentage of LA volume change -28.5+/-17.7 vs -20.5+/-20.0, p=0.019). In conclusion, on stepwise multiple regression analysis, preoperative LA volume, predominant lesion, age, and cardiac rhythm were significant predictors of LA reverse remodeling. A larger preoperative LA volume, MR rather than MS, younger age at the time of surgery, and sinus rhythm were important predictors of LA reverse remodeling after MV surgery.