PEG modification increases thermostability and inhibitor resistance of Bst DNA polymerase.

Polyethylene glycol modification (PEGylation) is a widely used strategy to improve the physicochemical properties of various macromolecules, especially protein drugs. However, its application in enhancing the performance of enzymes for molecular biology remains underexplored. This study explored the PEGylation of Bst DNA polymerase, determining optimal modification reaction conditions. In comparison to the unmodified wild-type counterpart, the modified Bst DNA polymerase exhibited significantly improved activity, thermal stability, and inhibitor tolerance during loop-mediated isothermal amplification. When applied for the detection of Salmonella in crude samples, the modified enzyme demonstrated a notably accelerated reaction rate. Therefore, PEGylation emerges as a viable strategy for refining DNA polymerases, helping in the development of novel molecular diagnostic reagents.

Improvement of duplex-specific nuclease salt tolerance by fusing DNA-binding domain of DNase from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix.

Salt tolerance is an important property of duplex-specific nuclease (DSN). DSN with high salt tolerance can be more widely used in genetic engineering, especially in the production of nucleic acid drugs. To improve the salt tolerance of DSN, we selected five DNA-binding domains from extremophilic organisms, which have been shown the ability to improve salt tolerance of DNA polymerases and nucleases. The experimental results demonstrated that the fusion protein TK-DSN produced by fusing a N-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs domain from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix, has a significantly improved salt tolerance. TK-DSN can tolerate the concentration of NaCl up to 800 mM; in addition, the ability of digesting DNA was also enhanced during in vitro transcription and RNA purification. This strategy provides the method for the personalized customization of biological tool enzymes for different applications.

Generation and Characterization of Monoclonal Antibodies Against Tth DNA Polymerase and its Application to Hot-Start PCR.

BACKGROUND: As a heat-resistant polymerase, Thermus thermophilus (Tth) DNA polymerase can be widely used in Polymerase Chain Reaction (PCR). However, its non-specific amplification phenomenon is serious, which greatly limits development. OBJECTIVE: In this study, we prepared Tth monoclonal antibodies against Tth DNA polymerase and researched their application in hot-start PCR. METHODS: Tth was recombinantly expressed and purified, and used as an antigen to immunize BALB/ c mice to obtain monoclonal antibodies. The qualified monoclonal antibody and Tth were incubated for a period of time at a certain temperature to obtain the hot-start Tth. We tested the polymerase activity and exonuclease activity blocking the performance of hot-start Tth. Finally, the hot-start Tth was applied to one-step RT-PCR. RESULTS: Tth with a purity of >95% was obtained, and ten monoclonal antibodies were obtained by immunization. After incubation, three monoclonal antibodies were identified that could inhibit the polymerase activity of Tth at low temperature. Furthermore, these three antibodies successfully eliminated non-specific amplification in practical applications. CONCLUSION: Three monoclonal antibodies were successfully validated. Among them, monoclonal antibody 9 had the best overall effect. They possess the function of inhibiting at low temperature and releasing at high temperature, which can be used as Tth polymerase inhibitors in the field of molecular diagnostics.

Auxin abolishes SHI-RELATED SEQUENCE5-mediated inhibition of lateral root development in Arabidopsis.

Lateral roots (LRs), which form in the plant postembryonically, determine the architecture of the root system. While negative regulatory factors that inhibit LR formation and are counteracted by auxin exist in the pericycle, these factors have not been characterised. Here, we report that SHI-RELATED SEQUENCE5 (SRS5) is an intrinsic negative regulator of LR formation and that auxin signalling abolishes this inhibitory effect of SRS5. Whereas LR primordia (LRPs) and LRs were fewer and less dense in SRS5ox and Pro35S:SRS5-GFP plants than in the wild-type, they were more abundant and denser in the srs5-2 loss-of-function mutant. SRS5 inhibited LR formation by directly downregulating the expression of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16) and LBD29. Auxin repressed SRS5 expression. Auxin-mediated repression of SRS5 expression was not observed in the arf7-1 arf19-1 double mutant, likely because ARF7 and ARF19 bind to the promoter of SRS5 and inhibit its expression in response to auxin. Taken together, our data reveal that SRS5 negatively regulates LR formation by repressing the expression of LBD16 and LBD29 and that auxin releases this inhibitory effect through ARF7 and ARF19.

Solution structure of SHIP2 SH2 domain and its interaction with a phosphotyrosine peptide from c-MET.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners.

The COP1 Target SHI-RELATED SEQUENCE5 Directly Activates Photomorphogenesis-Promoting Genes.

Plant seedlings undergo distinct developmental processes in the dark and in the light. Several genes, including ELONGATED HYPOCOTYL5 (HY5), B-BOX PROTEIN21 (BBX21), and BBX22, have been identified as photomorphogenesis-promoting factors in Arabidopsis thaliana; however, the overexpression of these genes does not induce photomorphogenesis in the dark. Using an activation-tagging approach, we identified SRS5ox, which overexpresses SHI-RELATED SEQUENCE5 (SRS5) following induction with estradiol. SRS5 overexpression in SRS5ox and Pro35S:SRS5-GFP seedlings results in a constitutive photomorphogenesis phenotype in the dark, whereas SRS5 loss of function in the srs5-2 mutant results in long hypocotyls in the light. This indicates that SRS5 is a positive regulator of photomorphogenesis. Furthermore, SRS5 promotes photomorphogenesis by directly binding to the promoters of photomorphogenesis-promoting genes, such as HY5, BBX21, and BBX22, and activating their expression, thus affecting the expression of downstream light-signaling genes. These data indicate that SRS5 acts in the upregulation of photomorphogenesis-promoting genes. In addition, CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which plays a central repressive role in seedling photomorphogenesis, directly ubiquitinates SRS5, promoting its degradation in the dark. Taken together, our results demonstrate that SRS5 directly activates the expression of downstream genes HY5, BBX21, and BBX22 and is a target of COP1-mediated degradation in Arabidopsis.

Histone H3 N-terminal acetylation sites especially K14 are important for rDNA silencing and aging.

Histone variants and histone modifications are essential components in the establishment and maintenance of the repressed status of heterochromatin. Among these histone variants and modifications, acetylation at histone H4K16 is uniquely important for the maintenance of silencing at telomere and mating type loci but not at the ribosomal DNA locus. Here we show that mutations at H3 N-terminal acetylation site K14 specifically disrupt rDNA silencing. However, the mutant ion at H3K14R doesn't affect the recruitment of Pol II repressor RENT (regulator of nucleolar silencing and telophase exit) complex at the rDNA region. Instead, the CAF-1(chromatin assembly factor I) subunit Cac2 level decreased in the H3K14R mutant. Further experiments revealed that the single mutation at H3K14 and multi-site mutations at H3 N-terminus including K14 also delayed replication-depend nucleosome assembly and advanced replicative life span. In conclusion, our data suggest that histone H3 N-terminal acetylation sites especially at K14 are important for rDNA silencing and aging.

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in Arabidopsis.

KEY MESSAGE: Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis. Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two ß subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1-CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

Ethylene Inhibits Root Elongation during Alkaline Stress through AUXIN1 and Associated Changes in Auxin Accumulation.

Soil alkalinity causes major reductions in yield and quality of crops worldwide. The plant root is the first organ sensing soil alkalinity, which results in shorter primary roots. However, the mechanism underlying alkaline stress-mediated inhibition of root elongation remains to be further elucidated. Here, we report that alkaline conditions inhibit primary root elongation of Arabidopsis (Arabidopsis thaliana) seedlings by reducing cell division potential in the meristem zones and that ethylene signaling affects this process. The ethylene perception antagonist silver (Ag(+)) alleviated the inhibition of root elongation by alkaline stress. Moreover, the ethylene signaling mutants ethylene response1-3 (etr1-3), ethylene insensitive2 (ein2), and ein3-1 showed less reduction in root length under alkaline conditions, indicating a reduced sensitivity to alkalinity. Ethylene biosynthesis also was found to play a role in alkaline stress-mediated root inhibition; the ethylene overproducer1-1 mutant, which overproduces ethylene because of increased stability of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE5, was hypersensitive to alkaline stress. In addition, the ethylene biosynthesis inhibitor cobalt (Co(2+)) suppressed alkaline stress-mediated inhibition of root elongation. We further found that alkaline stress caused an increase in auxin levels by promoting expression of auxin biosynthesis-related genes, but the increase in auxin levels was reduced in the roots of the etr1-3 and ein3-1 mutants and in Ag(+)/Co(2+)-treated wild-type plants. Additional genetic and physiological data showed that AUXIN1 (AUX1) was involved in alkaline stress-mediated inhibition of root elongation. Taken together, our results reveal that ethylene modulates alkaline stress-mediated inhibition of root growth by increasing auxin accumulation by stimulating the expression of AUX1 and auxin biosynthesis-related genes.

Low temperature inhibits root growth by reducing auxin accumulation via ARR1/12.

Plants exhibit reduced root growth when exposed to low temperature; however, how low temperature modulates root growth remains to be understood. Our study demonstrated that low temperature reduces both meristem size and cell number, repressing the division potential of meristematic cells by reducing auxin accumulation, possibly through the repressed expression of PIN1/3/7 and auxin biosynthesis-related genes, although the experiments with exogenous auxin application also suggest the involvement of other factor(s). In addition, we verified that ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12 are involved in low temperature-mediated inhibition of root growth by showing that the roots of arr1-3 arr12-1 seedlings were less sensitive than wild-type roots to low temperature, in terms of changes in root length and meristem cell number. Furthermore, low temperature reduced the levels of PIN1/3 transcripts and the auxin level to a lesser extent in arr1-3 arr12-1 roots than in wild-type roots, suggesting that cytokinin signaling is involved in the low-temperature-mediated reduction of auxin accumulation. Taken together, our data suggest that low temperature inhibits root growth by reducing auxin accumulation via ARR1/12.

GPCR crystallization using lipidic cubic phase technique.

G protein-coupled receptors (GPCRs), a family of seven-transmembrane receptors, are among the most important drug targets with over half of all marketed drugs targeting the family. However, only a handful of easily druggable GPCRs are successfully targeted by pharmaceuticals. Efforts to shift this intensive focus to other, more recalcitrant GPCR targets will increasingly draw on new information such as structural details, which have until recently proven tremendously challenging to gather for this class of protein receptors due to the difficulties in obtaining diffraction-quality crystals. Recently, the development and application of lipidic cubic phase (LCP) technology has reduced one major hurdle for crystallization of GPCRs, with 22 unique receptors being structurally characterized from LCP grown crystals over the span of seven years. This review focuses on the technological improvements for LCP that have led to its successful utilization on the GPCR family, including the most recent combination of LCP with the X-ray free-electron laser that dramatically reduces requirements on crystal size, and holds significant promise for shortening timelines for structure determination and for accessing previously unattainable structures such as those of signaling complexes.

Proper PIN1 distribution is needed for root negative phototropism in Arabidopsis.

Plants can be adapted to the changing environments through tropic responses, such as light and gravity. One of them is root negative phototropism, which is needed for root growth and nutrient absorption. Here, we show that the auxin efflux carrier PIN-FORMED (PIN) 1 is involved in asymmetric auxin distribution and root negative phototropism. In darkness, PIN1 is internalized and localized to intracellular compartments; upon blue light illumination, PIN1 relocalize to basal plasma membrane in root stele cells. The shift of PIN1 localization induced by blue light is involved in asymmetric auxin distribution and root negative phototropic response. Both blue-light-induced PIN1 redistribution and root negative phototropism is mediated by a BFA-sensitive trafficking pathway and the activity of PID/PP2A. Our results demonstrate that blue-light-induced PIN1 redistribution participate in asymmetric auxin distribution and root negative phototropism.

Glucose inhibits root meristem growth via ABA INSENSITIVE 5, which represses PIN1 accumulation and auxin activity in Arabidopsis.

Glucose functions as a hormone-like signalling molecule that modulates plant growth and development in Arabidopsis thaliana. However, the role of glucose in root elongation remains elusive. Our study demonstrates that high concentrations of glucose reduce the size of the root meristem zone by repressing PIN1 accumulation and thereby reducing auxin levels. In addition, we verified the involvement of ABA INSENSITIVE 5 (ABI5) in this process by showing that abi5-1 is less sensitive to glucose than the wild type, whereas glucose induces ABI5 expression and the inducible overexpression of ABI5 reduces the size of the root meristem zone. Furthermore, the inducible overexpression of ABI5 in PIN1::PIN1-GFP plants reduces the level of PIN1-GFP, but glucose reduces the level of PIN1-GFP to a lesser extent in abi5-1 PIN1::PIN1-GFP plants than in the PIN1::PIN1-GFP control, suggesting that ABI5 is involved in glucose-regulated PIN1 accumulation. Taken together, our data suggest that ABI5 functions in the glucose-mediated inhibition of the root meristem zone by repressing PIN1 accumulation, thus leading to reduced auxin levels in roots.

Blue-light-induced PIN3 polarization for root negative phototropic response in Arabidopsis.

Root negative phototropism is an important response in plants. Although blue light is known to mediate this response, the cellular and molecular mechanisms underlying root negative phototropism remain unclear. Here, we report that the auxin efflux carrier PIN-FORMED (PIN) 3 is involved in asymmetric auxin distribution and root negative phototropism. Unilateral blue-light illumination polarized PIN3 to the outer lateral membrane of columella cells at the illuminated root side, and increased auxin activity at the illuminated side of roots, where auxin promotes growth and causes roots bending away from the light source. Furthermore, root negative phototropic response and blue-light-induced PIN3 polarization were modulated by a brefeldin A-sensitive, GNOM-dependent, trafficking pathway and by phot1-regulated PINOID (PID)/PROTEIN PHOSPHATASE 2A (PP2A) activity. Our results indicate that blue-light-induced PIN3 polarization is needed for asymmetric auxin distribution during root negative phototropic response.

Copper regulates primary root elongation through PIN1-mediated auxin redistribution.

The heavy metal copper (Cu) is an essential microelement required for normal plant growth and development, but it inhibits primary root growth when in excess. The mechanism underlying how excess Cu functions in this process remains to be further elucidated. Here, we report that a higher concentration of CuSO4 inhibited primary root elongation of Arabidopsis seedlings by affecting both the elongation and meristem zones. In the meristem zone, meristematic cell division potential was reduced by excess Cu. Further experiments showed that Cu can modulate auxin distribution, resulting in higher auxin activities in both the elongation and meristem zones of Cu-treated roots based on DR5::GUS expression patterns. This Cu-mediated auxin redistribution was shown to be responsible for Cu-mediated inhibition of primary root elongation. Additional genetic and physiological data demonstrated that it was PINFORMED1 (PIN1), but not PIN2 or AUXIN1 (AUX1), that regulated this process. However, Cu-induced hydrogen peroxide accumulation did not contribute to Cu-induced auxin redistribution for inhibition of root elongation. When the possible role of ethylene in this process was analyzed, Cu had a similar impact on the root elongation of both the wild type and the ein2-1 mutant, implying that Cu-mediated inhibition of primary root elongation was not due to the ethylene signaling pathway.

The Bphi008a gene interacts with the ethylene pathway and transcriptionally regulates MAPK genes in the response of rice to brown planthopper feeding.

We examined ways in which the Brown planthopper induced008a (Bphi008a; AY256682) gene of rice (Oryza sativa) enhances the plant's resistance to a specialist herbivore, the brown planthopper (BPH; Nilaparvata lugens). Measurement of the expression levels of ethylene synthases and of ethylene emissions showed that BPH feeding rapidly initiated the ethylene signaling pathway and up-regulated Bphi008a transcript levels after 6 to 96 h of feeding. In contrast, blocking ethylene transduction (using 1-methylcyclopropene) reduced Bphi008a transcript levels in wild-type plants fed upon by BPH. In vitro kinase assays showed that Bphi008a can be phosphorylated by rice Mitogen-activated Protein Kinase5 (OsMPK5), and yeast two-hybrid assays demonstrated that the carboxyl-terminal proline-rich region of Bphi008a interacts directly with this kinase. Furthermore, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus. Subsequently, we found that Bphi008a up-regulation and down-regulation were accompanied by different changes in transcription levels of OsMPK5, OsMPK12, OsMPK13, and OsMPK17 in transgenic plants. Immunoblot analysis also showed that the OsMPK5 protein level increased in overexpressing plants and decreased in RNA interference plants after BPH feeding. In transgenic lines, changes in the expression levels of several enzymes that are important components of the defenses against the BPH were also observed. Finally, yeast two-hybrid screening results showed that Bphi008a is able to interact with a b-ZIP transcription factor (OsbZIP60) and a RNA polymerase polypeptide (SDRP).

Improvement of growth and camptothecin yield by altering nitrogen source supply in cell suspension cultures of Camptotheca acuminata.

Nitrate at 70 mM gave the highest biomass of Camptotheca acuminata in suspension culture in MS medium, but a NH4+/NO3- molar ratio of 5:1 (giving a total of 40 mM N) gave the maximum camptothecin yield. A two-stage flask culture system was established to improve culture efficiency; cell dry weight, camptothecin content and yield was increased by 30%, 280% and 340%, respectively when compared with those of control, reaching up to 36 g l(-1), 0.36 mg g(-1), and 12.8 mg l(-1), respectively.