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

Design, Modeling, and Analysis of a Novel Hydraulic Energy-Regenerative Shock Absorber for Vehicle Suspension

To reduce energy consumption or improve energy efficiency, the regenerative devices recently have drawn the public’s eyes. In this paper, a novel hydraulic energy-regenerative shock absorber (HERSA) is developed for vehicle suspension to regenerate the vibration energy which is dissipated by conventional viscous dampers into heat waste. At first, the schematic of HERSA is presented and a mathematic model is developed to describe the characteristic of HERSA. Then the parametric sensitivity analysis of the vibration energy is expounded, and the ranking of their influences is k1≫m2>m1>k2≈cs. Besides, a parametric study of HERSA is adopted to research the influences of the key parameters on the characteristic of HERSA. Moreover, an optimization of HERSA is carried out to regenerate more power as far as possible without devitalizing the damping characteristic. To make the optimization results more close to the actual condition, the displacement data of the shock absorber in the road test is selected as the excitation in the optimization. The results show that the RMS of regenerated energy is up to 107.94 W under the actual excitation. Moreover it indicates that the HERSA can improve its performance through the damping control

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. 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A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta. Plant Physiology, 133(2), 462-469. doi:10.1104/pp.103.027979Cutler, S. R., Rodriguez, P. L., Finkelstein, R. R., & Abrams, S. R. (2010). Abscisic Acid: Emergence of a Core Signaling Network. Annual Review of Plant Biology, 61(1), 651-679. doi:10.1146/annurev-arplant-042809-112122Edel, K. H., & Kudla, J. (2016). Integration of calcium and ABA signaling. Current Opinion in Plant Biology, 33, 83-91. doi:10.1016/j.pbi.2016.06.010French, A. P., Mills, S., Swarup, R., Bennett, M. J., & Pridmore, T. P. (2008). Colocalization of fluorescent markers in confocal microscope images of plant cells. Nature Protocols, 3(4), 619-628. doi:10.1038/nprot.2008.31Gehl, C., Waadt, R., Kudla, J., Mendel, R.-R., & Hänsch, R. (2009). New GATEWAY vectors for High Throughput Analyses of Protein–Protein Interactions by Bimolecular Fluorescence Complementation. Molecular Plant, 2(5), 1051-1058. doi:10.1093/mp/ssp040Herranz, M. C., Pallas, V., & Aparicio, F. (2012). Multifunctional Roles for the N-Terminal Basic Motif of Alfalfa mosaic virus Coat Protein: Nucleolar/Cytoplasmic Shuttling, Modulation of RNA-Binding Activity, and Virion Formation. Molecular Plant-Microbe Interactions®, 25(8), 1093-1103. doi:10.1094/mpmi-04-12-0079-rHornáček, M., Kováčik, L., Mazel, T., Cmarko, D., Bártová, E., Raška, I., & Smirnov, E. (2017). Fluctuations of pol I and fibrillarin contents of the nucleoli. Nucleus, 8(4), 421-432. doi:10.1080/19491034.2017.1306160Irigoyen, M. L., Iniesto, E., Rodriguez, L., Puga, M. I., Yanagawa, Y., Pick, E., … Rubio, V. (2014). Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis. The Plant Cell, 26(2), 712-728. doi:10.1105/tpc.113.122234Kong, L., Cheng, J., Zhu, Y., Ding, Y., Meng, J., Chen, Z., … Gong, Z. (2015). 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. The Plant Cell, 19(9), 2804-2821. doi:10.1105/tpc.107.051870Pizzio, G. A., Rodriguez, L., Antoni, R., Gonzalez-Guzman, M., Yunta, C., Merilo, E., … Rodriguez, P. L. (2013). The PYL4 A194T Mutant Uncovers a Key Role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA Interaction for Abscisic Acid Signaling and Plant Drought Resistance      . Plant Physiology, 163(1), 441-455. doi:10.1104/pp.113.224162Rodriguez, L., Gonzalez-Guzman, M., Diaz, M., Rodrigues, A., Izquierdo-Garcia, A. C., Peirats-Llobet, M., … Rodriguez, P. L. (2014). C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis. The Plant Cell, 26(12), 4802-4820. doi:10.1105/tpc.114.129973Romero-Barrios, N., & Vert, G. (2017). Proteasome-independent functions of lysine-63 polyubiquitination in plants. New Phytologist, 217(3), 995-1011. doi:10.1111/nph.14915Rubio, S., Rodrigues, A., Saez, A., Dizon, M. B., Galle, A., Kim, T.-H., … Rodriguez, P. L. (2009). Triple Loss of Function of Protein Phosphatases Type 2C Leads to Partial Constitutive Response to Endogenous Abscisic Acid      . Plant Physiology, 150(3), 1345-1355. doi:10.1104/pp.109.137174Saez, A., Rodrigues, A., Santiago, J., Rubio, S., & Rodriguez, P. L. (2008). HAB1–SWI3B Interaction Reveals a Link between Abscisic Acid Signaling and Putative SWI/SNF Chromatin-Remodeling Complexes in Arabidopsis. The Plant Cell, 20(11), 2972-2988. doi:10.1105/tpc.107.056705Santiago, J., Rodrigues, A., Saez, A., Rubio, S., Antoni, R., Dupeux, F., … Rodriguez, P. L. (2009). Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. The Plant Journal, 60(4), 575-588. doi:10.1111/j.1365-313x.2009.03981.xSantiago, J., Dupeux, F., Round, A., Antoni, R., Park, S.-Y., Jamin, M., … Márquez, J. A. (2009). The abscisic acid receptor PYR1 in complex with abscisic acid. Nature, 462(7273), 665-668. doi:10.1038/nature08591Schapire, A. L., Voigt, B., Jasik, J., Rosado, A., Lopez-Cobollo, R., Menzel, D., … Botella, M. A. (2008). Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability. The Plant Cell, 20(12), 3374-3388. doi:10.1105/tpc.108.063859Sheen, J. (1998). Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants. Proceedings of the National Academy of Sciences, 95(3), 975-980. doi:10.1073/pnas.95.3.975Tischer, S. V., Wunschel, C., Papacek, M., Kleigrewe, K., Hofmann, T., Christmann, A., & Grill, E. (2017). Combinatorial interaction network of abscisic acid receptors and coreceptors fromArabidopsis thaliana. Proceedings of the National Academy of Sciences, 114(38), 10280-10285. doi:10.1073/pnas.1706593114Turnbull, D., & Hemsley, P. A. (2017). Fats and function: protein lipid modifications in plant cell signalling. Current Opinion in Plant Biology, 40, 63-70. doi:10.1016/j.pbi.2017.07.007Umezawa, T., Sugiyama, N., Mizoguchi, M., Hayashi, S., Myouga, F., Yamaguchi-Shinozaki, K., … Shinozaki, K. (2009). Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proceedings of the National Academy of Sciences, 106(41), 17588-17593. doi:10.1073/pnas.0907095106Vlad, F., Rubio, S., Rodrigues, A., Sirichandra, C., Belin, C., Robert, N., … Merlot, S. (2009). Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid inArabidopsis . The Plant Cell, 21(10), 3170-3184. doi:10.1105/tpc.109.069179Wu, C., Feng, J., Wang, R., Liu, H., Yang, H., Rodriguez, P. L., … Wang, D. (2012). HRS1 Acts as a Negative Regulator of Abscisic Acid Signaling to Promote Timely Germination of Arabidopsis Seeds. 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Current evidence, clinical applications, and future directions of transcranial magnetic stimulation as a treatment for ischemic stroke

Transcranial magnetic stimulation (TMS) is a non-invasive brain neurostimulation technique that can be used as one of the adjunctive treatment techniques for neurological recovery after stroke. Animal studies have shown that TMS treatment of rats with middle cerebral artery occlusion (MCAO) model reduced cerebral infarct volume and improved neurological dysfunction in model rats. In addition, clinical case reports have also shown that TMS treatment has positive neuroprotective effects in stroke patients, improving a variety of post-stroke neurological deficits such as motor function, swallowing, cognitive function, speech function, central post-stroke pain, spasticity, and other post-stroke sequelae. However, even though numerous studies have shown a neuroprotective effect of TMS in stroke patients, its possible neuroprotective mechanism is not clear. Therefore, in this review, we describe the potential mechanisms of TMS to improve neurological function in terms of neurogenesis, angiogenesis, anti-inflammation, antioxidant, and anti-apoptosis, and provide insight into the current clinical application of TMS in multiple neurological dysfunctions in stroke. Finally, some of the current challenges faced by TMS are summarized and some suggestions for its future research directions are made

Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in rice

Pre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), ζ-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene β-cyclase (β-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1 mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice

Authenticating the geographic origins of Atractylodes lancea rhizome chemotypes in China through metabolite marker identification

IntroductionAtractylodes lancea is widely distributed in East Asia, ranging from Amur to south-central China. The rhizome of A. lancea is commonly used in traditional Chinese medicine, however, the quality of products varies across different regions with different geochemical characteristics.MethodThis study aimed to identify the chemotypes of A. lancea from different areas and screen for chemical markers by quantifying volatile organic compounds (VOCs) using a targeted metabolomics approach based on GC–MS/MS.ResultsThe A. lancea distributed in Hubei, Anhui, Shaanxi, and a region west of Henan province was classified as the Hubei Chemotype (HBA). HBA is characterized by high content of β-eudesmol and hinesol with lower levels of atractylodin and atractylon. In contrast, the Maoshan Chemotype (MA) from Jiangsu, Shandong, Shanxi, Hebei, Inner Mongolia, and other northern regions, exhibited high levels of atractylodin and atractylon. A total of 15 categories of VOCs metabolites were detected and identified, revealing significant differences in the profiles of terpenoid, heterocyclic compound, ester, and ketone among different areas. Multivariate statistics indicated that 6 compounds and 455 metabolites could serve as candidate markers for differentiating A. lancea obtained from the southern, northern, and Maoshan areas.DiscussionThis comprehensive analysis provides a chemical fingerprint of selected A. lancea. Our results highlight the potential of metabolite profiling combined with chemometrics for authenticating the geographical origin of A. lancea

Insights into salt tolerance from the genome of Thellungiella salsuginea

Thellungiella salsuginea, a close relative of Arabidopsis, represents an extremophile model for abiotic stress tolerance studies. We present the draft sequence of the T. salsuginea genome, assembled based on ∼134-fold coverage to seven chromosomes with a coding capacity of at least 28,457 genes. This genome provides resources and evidence about the nature of defense mechanisms constituting the genetic basis underlying plant abiotic stress tolerance. Comparative genomics and experimental analyses identified genes related to cation transport, abscisic acid signaling, and wax production prominent in T. salsuginea as possible contributors to its success in stressful environments

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