SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples

Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA

[EN] Abscisic acid (ABA) is an essential hormone for plant development and stress responses. ABA signaling is suppressed by clade A PP2C phosphatases, which function as key repressors of this pathway through inhibiting ABA-activated SnRK2s (SNF1-related protein kinases). Upon ABA perception, the PYR/PYL/RCAR ABA receptors bind to PP2Cs with high affinity and biochemically inhibit their activity. While thismechanismhas been extensively studied, how PP2Cs are regulated at the protein level is only starting to be explored. Arabidopsis thaliana RING DOMAIN LIGASE5 (RGLG5) belongs to a five-member E3 ubiquitin ligase family whose target proteins remain unknown. We report that RGLG5, together with RGLG1, releases the PP2C blockade of ABA signaling by mediating PP2CA protein degradation. ABA promotes the interaction of PP2CA with both E3 ligases, which mediate ubiquitination of PP2CA and are required for ABA-dependent PP2CA turnover. Downregulation of RGLG1 and RGLG5 stabilizes endogenous PP2CA and diminishes ABA-mediated responses. Moreover, the reduced response to ABA in germination assays is suppressed in the rglg1 amiR (artificial microRNA)-rglg5 pp2ca-1 triple mutant, supporting a functional link among these loci. Overall, our data indicate that RGLG1 and RGLG5 are important modulators of ABA signaling, and they unveil amechanismfor activation of the ABA pathway by controlling PP2C half-life.We thank Andreas Bachmair for the rglg1 mutant, Sean R. Cutler for the pyr1 pyl1 pyl2 pyl4 seeds, Dapeng Zhang for the transgenic material harboring ABI2, Hongwei Guo and Jianmin Zhou for the pCAMBIA1300-Nluc and pCAMBIA1300-Cluc vectors, and John Olson for assistance in English editing. Work in C.A.'s laboratory was supported by grants from the National Key Basic Science "973" Program (Grant 2012CB114006), the National Natural Science Foundation (Grants 31272023, 31170231, and 90817001) of the Chinese government, and by the State Key Laboratory of Protein and Plant Gene Research, Peking University. Work in P.L.R.'s laboratory was supported by Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (Grant BIO2014-52537-R).Wu, Q.; Zhang, X.; Peirats-Llobet, M.; Belda Palazón, B.; Wang, X.; Cui, S.; Yu, X.... (2016). Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA. Plant Cell. 28(9):2178-2196. https://doi.org/10.1105/tpc.16.003642178219628

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

[EN] Hormone- and stress-induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone-induced ubiquitination plays a crucial role to determine half-life of key negative regulators of hormone signaling. For ABA signaling, degradation of clade A PP2Cs, such as PP2CA or ABI1, is a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. ABA promotes the degradation of PP2CA through the RGLG1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG1 with PP2CA given they are predominantly found in plasma membrane and nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG1 and promotes nuclear interaction with PP2CA. We found RGLG1 is myristoylated in vivo, which facilitates its attachment to plasma membrane. ABA inhibits myristoylation of RGLG1 through downregulation of Nmyristoyltransferase1 (NMT1) and promotes nuclear translocation of RGLG1 in a cycloheximide-insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CA protein levels and the formation of RGLG1-receptor-phosphatase complexes. We show that RGLG1Gly2Ala -mutated in the Nterminal myristoylation site- shows constitutive nuclear localization and causes enhanced response to ABA and salt/osmotic stress. RGLG1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG1-PP2CA-PYL8. In summary, we provide evidence that an E3 ligase can dynamically re-localize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG1-PP2CA interaction and hence PP2CA degradation.Work in P.L.R.'s laboratory was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas through grants BIO2014-52537-R and BIO2017-82503-R. This work was also funded by grants from the Deutsche Forschungsgemeinschaft (DFG) Ku931/4-1 to J. K., and BA4742/1-2 to O.B. B.B. was funded by Programa VALi+ d GVA APOSTD/2017/039. J.J. was supported by an FPI contract from MINECOBelda Palazón, B.; Julian, J.; Coego, A.; Wu, Q.; Zhang, X.; Batistic, O.; Alquraishi, SA.... (2019). ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA. The Plant Journal. 98(5):813-825. https://doi.org/10.1111/tpj.14274S813825985Antoni, R., Gonzalez-Guzman, M., Rodriguez, L., Rodrigues, A., Pizzio, G. A., & Rodriguez, P. L. (2011). Selective Inhibition of Clade A Phosphatases Type 2C by PYR/PYL/RCAR Abscisic Acid Receptors    . Plant Physiology, 158(2), 970-980. doi:10.1104/pp.111.188623Antoni, R., Gonzalez-Guzman, M., Rodriguez, L., Peirats-Llobet, M., Pizzio, G. A., Fernandez, M. A., … Rodriguez, P. L. (2012). PYRABACTIN RESISTANCE1-LIKE8 Plays an Important Role for the Regulation of Abscisic Acid Signaling in Root      . Plant Physiology, 161(2), 931-941. doi:10.1104/pp.112.208678Belda-Palazon, B., Gonzalez-Garcia, M.-P., Lozano-Juste, J., Coego, A., Antoni, R., Julian, J., … Rodriguez, P. L. (2018). PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization–based mechanisms. Proceedings of the National Academy of Sciences, 115(50), E11857-E11863. doi:10.1073/pnas.1815410115Bhaskara, G. 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Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases. Nature Communications, 6(1). doi:10.1038/ncomms9630Kuhn, J. M., Boisson-Dernier, A., Dizon, M. B., Maktabi, M. H., & Schroeder, J. I. (2005). The Protein Phosphatase AtPP2CA Negatively Regulates Abscisic Acid Signal Transduction in Arabidopsis, and Effects of abh1 on AtPP2CA mRNA  . Plant Physiology, 140(1), 127-139. doi:10.1104/pp.105.070318Lee, S. C., Lan, W., Buchanan, B. B., & Luan, S. (2009). A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells. Proceedings of the National Academy of Sciences, 106(50), 21419-21424. doi:10.1073/pnas.0910601106Lee, H.-J., Park, Y.-J., Seo, P. J., Kim, J.-H., Sim, H.-J., Kim, S.-G., & Park, C.-M. (2015). Systemic Immunity Requires SnRK2.8-Mediated Nuclear Import of NPR1 in Arabidopsis. The Plant Cell, 27(12), 3425-3438. doi:10.1105/tpc.15.00371Leitner, J., Petrasek, J., Tomanov, K., Retzer, K., Parezova, M., Korbei, B., … Luschnig, C. (2012). Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth. Proceedings of the National Academy of Sciences, 109(21), 8322-8327. doi:10.1073/pnas.1200824109Li, W., & Schmidt, W. (2010). A lysine-63-linked ubiquitin chain-forming conjugase, UBC13, promotes the developmental responses to iron deficiency in Arabidopsis roots. The Plant Journal, 62(2), 330-343. doi:10.1111/j.1365-313x.2010.04150.xLumba, S., Cutler, S., & McCourt, P. (2010). Plant Nuclear Hormone Receptors: A Role for Small Molecules in Protein-Protein Interactions. Annual Review of Cell and Developmental Biology, 26(1), 445-469. doi:10.1146/annurev-cellbio-100109-103956Lynch, T., Erickson, B. J., & Finkelstein, R. R. (2012). Direct interactions of ABA-insensitive(ABI)-clade protein phosphatase(PP)2Cs with calcium-dependent protein kinases and ABA response element-binding bZIPs may contribute to turning off ABA response. Plant Molecular Biology, 80(6), 647-658. doi:10.1007/s11103-012-9973-3Majeran, W., Le Caer, J.-P., Ponnala, L., Meinnel, T., & Giglione, C. (2018). Targeted Profiling of Arabidopsis thaliana Subproteomes Illuminates Co- and Posttranslationally N-Terminal Myristoylated Proteins. The Plant Cell, 30(3), 543-562. doi:10.1105/tpc.17.00523Moes, D., Himmelbach, A., Korte, A., Haberer, G., & Grill, E. (2008). Nuclear localization of the mutant protein phosphatase abi1 is required for insensitivity towards ABA responses in Arabidopsis. The Plant Journal, 54(5), 806-819. doi:10.1111/j.1365-313x.2008.03454.xMoreno-Alvero, M., Yunta, C., Gonzalez-Guzman, M., Lozano-Juste, J., Benavente, J. L., Arbona, V., … Albert, A. (2017). Structure of Ligand-Bound Intermediates of Crop ABA Receptors Highlights PP2C as Necessary ABA Co-receptor. Molecular Plant, 10(9), 1250-1253. doi:10.1016/j.molp.2017.07.004NAKAGAWA, T., SUZUKI, T., MURATA, S., NAKAMURA, S., HINO, T., MAEO, K., … ISHIGURO, S. (2007). Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs in Transgenic Analysis of Plants. Bioscience, Biotechnology, and Biochemistry, 71(8), 2095-2100. doi:10.1271/bbb.70216Peirats-Llobet, M., Han, S.-K., Gonzalez-Guzman, M., Jeong, C. W., Rodriguez, L., Belda-Palazon, B., … Rodriguez, P. L. (2016). A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway Components. Molecular Plant, 9(1), 136-147. doi:10.1016/j.molp.2015.10.003Pierre, M., Traverso, J. A., Boisson, B., Domenichini, S., Bouchez, D., Giglione, C., & Meinnel, T. (2007). N-Myristoylation Regulates the SnRK1 Pathway inArabidopsis. 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The LSD1-Type Zinc Finger Motifs of Pisum sativa LSD1 Are a Novel Nuclear Localization Signal and Interact with Importin Alpha

Background: Genetic studies of the Arabidopsis mutant lsd1 highlight the important role of LSD1 in the negative regulation of plant programmed cell death (PCD). Arabidopsis thaliana LSD1 (AtLSD1) contains three LSD1-type zinc finger motifs, which are involved in the protein-protein interaction. Methodology/Principal Findings: To further understand the function of LSD1, we have analyzed cellular localization and functional localization domains of Pisum sativa LSD1 (PsLSD1), which is a homolog of AtLSD1. Subcellular localization analysis of green fluorescent protein (GFP)-tagged PsLSD1 indicates that PsLSD1 is localized in the nucleus. Using a series of GFP-tagged PsLSD1 deletion mutants, we found that the three LSD1-type zinc finger motifs of PsLSD1 alone can target GFP to the nucleus, whereas deletion of the three zinc finger motifs or any individual zinc finger motif causes PsLSD1 to lose its nuclear localization, indicating that the three zinc finger motifs are necessary and sufficient for its nuclear localization. Moreover, site-directed mutagenesis analysis of GFP-tagged PsLSD1 indicates that tertiary structure and basic amino acids of each zinc finger motif are necessary for PsLSD1 nuclear localization. In addition, yeast two-hybrid, pull-down, and BiFC assays demonstrate that the three zinc finger motifs of PsLSD1 directly bind to importin alpha in vitro and in vivo. Conclusions/Significance: Our data demonstrate that the LSD1-type zinc finger motifs of PsLSD1 are a novel nuclear localization signal and directly bind to importin alpha, and suggest that the nuclear import of LSD1 may rely on the interaction between its zinc finger motifs and importin alpha. Moreover, the nuclear localization of PsLSD1 suggests that LSD1 may function as a transcription regulator involved in negatively regulating PCD.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000292929500042&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Multidisciplinary SciencesSCI(E)PubMed11ARTICLE7e22131

The LSD1-Interacting Protein GILP Is a LITAF Domain Protein That Negatively Regulates Hypersensitive Cell Death in Arabidopsis

Hypersensitive cell death, a form of avirulent pathogen-induced programmed cell death (PCD), is one of the most efficient plant innate immunity. However, its regulatory mechanism is poorly understood. AtLSD1 is an important negative regulator of PCD and only two proteins, AtbZIP10 and AtMC1, have been reported to interact with AtLSD1.To identify a novel regulator of hypersensitive cell death, we investigate the possible role of plant LITAF domain protein GILP in hypersensitive cell death. Subcellular localization analysis showed that AtGILP is localized in the plasma membrane and its plasma membrane localization is dependent on its LITAF domain. Yeast two-hybrid and pull-down assays demonstrated that AtGILP interacts with AtLSD1. Pull-down assays showed that both the N-terminal and the C-terminal domains of AtGILP are sufficient for interactions with AtLSD1 and that the N-terminal domain of AtLSD1 is involved in the interaction with AtGILP. Real-time PCR analysis showed that AtGILP expression is up-regulated by the avirulent pathogen Pseudomonas syringae pv. tomato DC3000 avrRpt2 (Pst avrRpt2) and fumonisin B1 (FB1) that trigger PCD. Compared with wild-type plants, transgenic plants overexpressing AtGILP exhibited significantly less cell death when inoculated with Pst avrRpt2, indicating that AtGILP negatively regulates hypersensitive cell death.These results suggest that the LITAF domain protein AtGILP localizes in the plasma membrane, interacts with AtLSD1, and is involved in negatively regulating PCD. We propose that AtGILP functions as a membrane anchor, bringing other regulators of PCD, such as AtLSD1, to the plasma membrane. Human LITAF domain protein may be involved in the regulation of PCD, suggesting the evolutionarily conserved function of LITAF domain proteins in the regulation of PCD

Calibration of UWA’s O-tube flume facility

The O-tube facility, designed and established at the University of Western Australia, is an innovative closed loop flume in which a random storm sequence can be reproduced via control of a large pump system. The O-tube facility is capable of simulating hydrodynamic conditions near the seabed and the interaction with seabed sediment and any infrastructure that is resting on it.The purpose of carrying out the O-tube calibration described in this paper is to obtain the relationship between the motor rotation movement and the flow velocity generated in the O-tube, such that any required storm history within the performance envelope of the O-tube can be reproduced. A range of flow velocities and the corresponding pump speeds were measured under steady current, oscillatory flow and combined flow conditions. It was found that the relationship between the pump speed and the flow velocity varies with the oscillatory flow period. Based on the pump characteristic curves and O-tube system curves, the correlation between the motor speed and the flow velocity was derived by applying hydraulic theory and the principle of energy conservation.The derived correlation is validated by reproducing a wide range of target storm series, including a (1:5.8) scaled 100-year return period storm from the North West Shelf of Western Australia in 40 m water depth

A re-examination of the hydrodynamic forces acting on partially-buried submarine pipelines

The hydrodynamic forces exerted on a pipeline partially buried in a permeable seabed subjected to combined random oscillatory flow are investigated experimentally. The tests were carried out in a re-circulating flume that can generate steady currents, oscillatory flow and combined flow, known as the O-tube. The performance of the testing facility was validated against published experimental data under regular oscillatory flow conditions. Four tests with different embedment depths, under random oscillatory flow conditions were then carried out. The flow velocity, hydrodynamic pressure around the model pipe and pore pressure in the soil were monitored. The hydrodynamic forces were calculated through pressure integration around the model pipe. The hydrodynamic force reduction due to embedment was compared with the model given in the most widely-used pipeline stability design guideline, DNV-RPF109. A horizontal load reduction of 55% was found for the fully buried pipe, which was lower than the 70% reduction suggested by DNV-RP-F109. The variation of the vertical hydrodynamic load with embedment ratio (embedment depth to pipe diameter) was also found to be different from the model suggested by DNV-RP-F109. It was found the fully buried pipe experienced a large vertical force, even higher than that of the fully exposed pipe, under low KC number conditions.</p

A model for predicting pipeline sinkage induced by tunnel scour

The current design practice for subsea pipeline on-bottom stability (e.g. DNV-RP-F109) does not account the effect of sediment transport around a pipeline. Both field survey and small scale model test results show that seabed scour has a significant effect on pipeline embedment and therefore stability. Physical model tests carried out in an innovative large experimental facility, named the O-tube, at the University of Western Australia, have shown that tunnel scour and the subsequent pipe sinkage into the scour hole tend to stabilize a pipeline which might otherwise become unstable on an assumed stationary seabed, under ramping-up flow conditions. A simple calculation model that incorporates the three-dimensional scour and pipe sinkage due to the soil bearing capacity failure at the supporting span shoulders is proposed. The model parameters were calibrated using the O-tube experimental results. The model serves as a key element of a new pipeline stability analysis method that takes into account seabed mobility.</p

ABI4 Activates DGAT1 Expression in Arabidopsis Seedlings during Nitrogen Deficiency1[C][W][OA]

Triacylglycerol (TAG) is the major seed storage lipid and is important for biofuel and other renewable chemical uses. Acyl-coenzyme A:diacylglycerol acyltransferase1 (DGAT1) is the rate-limiting enzyme in the TAG biosynthesis pathway, but the mechanism of its regulation is unknown. Here, we show that TAG accumulation in Arabidopsis (Arabidopsis thaliana) seedlings increased significantly during nitrogen deprivation (0.1 mm nitrogen) with concomitant induction of genes involved in TAG biosynthesis and accumulation, such as DGAT1 and OLEOSIN1. Nitrogen-deficient seedlings were used to determine the key factors contributing to ectopic TAG accumulation in vegetative tissues. Under low-nitrogen conditions, the phytohormone abscisic acid plays a crucial role in promoting TAG accumulation in Arabidopsis seedlings. Yeast one-hybrid and electrophoretic mobility shift assays demonstrated that ABSCISIC ACID INSENSITIVE4 (ABI4), an important transcriptional factor in the abscisic acid signaling pathway, bound directly to the CE1-like elements (CACCG) present in DGAT1 promoters. Genetic studies also revealed that TAG accumulation and DGAT1 expression were reduced in the abi4 mutant. Taken together, our results indicate that abscisic acid signaling is part of the regulatory machinery governing TAG ectopic accumulation and that ABI4 is essential for the activation of DGAT1 in Arabidopsis seedlings during nitrogen deficiency

A new facility for studying ocean-structure-seabed interactions: The O-tube

This paper describes a unique new physical testing facility for studying ocean-structure-seabed interactions, and in particular pipeline on-bottom stability on erodible seabed under hydrodynamic loading. The facility, named the O-tube due to its shape, is a fully enclosed flume in which ambient and storm-induced near-seabed flows are generated by a computer-controlled flow pump. Combined steady and oscillatory flow can be generated by alternating pump flow directions in a controlled manner, and computer control also allows irregular flow to be generated. The design of the O-tube combines the capabilities of a conventional open channel flume (which provides steady current) with a U-tube (which provides oscillatory flow). The facility is designed to physically model severe storm conditions, as well as ambient or tidal flows. When studying pipeline stability on erodible seabed under severe hydrodynamic loading conditions, tests can be performed at a relatively large scale (typically 1/5) for large diameter pipelines (e.g. 40. in. gas trunklines) and at full scale for small diameter pipelines (&lt;. 8. in.) to minimize potential scaling effects associated with movable bed model tests. The specifications of the O-tube, a model pipe and an actuator system that supports the model pipe are given in detail. Preliminary model testing results show that the facility has met its design expectations.</p