Hormone receptor positive (HR+) breast cancer (BCa) is the most frequently diagnosed subtype. Acquired and intrinsic resistance to conventional endocrine therapy (ET) commonly occurs and prompts incurable metastatic disease. Hence, ET-resistant (ET-R) HR+ BCa presents a therapeutic challenge. Previous studies show elevated androgen receptor (AR) that supports resistance to ET tamoxifen and correlates with HR+ BCa metastasis. Yet surprisingly, studies with AR-blocker enzalutamide (Enz) in ET-R HR+ BCa present conflicting results. We now report that a constitutively active, unique from canonical Enz-targeted, AR accumulates in endocrine resistant HR+ BCa cells.AR protein profiles in acquired and intrinsic ET-R HR + -BCa were defined with cell-free modification tests, in-house in-vivo SUMOylation assays, and PLA imaging. Genomic activity of native AR and modified-AR mimetic was tested with reporter assays and limited transcriptome analysis. Spheroid growth and migration studies were used to evaluate inhibitory actions of Enz and combinatorial therapy.Sustained higher molecular weight SUMO-modified AR (SUMO-AR) persists in acquired and intrinsic ET-R BCa cell lines. Concurrently, SUMO isoforms and global SUMO-modified proteome also accumulates in the same cell lines. We identified AR as a novel substrate for the SUMO-E3 ligase HSPB1/Hsp27. Independent of ligand, SUMO-AR is resilient to ubiquitin-mediated proteasomal degradation, enriched in the nucleus, readily chromatin-bound, and transcriptionally active. Constitutive SUMO-AR initiates a gene-expression profile that favors epithelial-mesenchymal transition. Enz combined with a SUMO inhibitor attenuates migration and metastatic phenotype of ET-R HR+ BCa.Targeting both unmodified and SUMO-modified AR prevents the metastatic progression of HR+ BCa with ET-R.Video abstract Endocrine therapy (ET) remains the first line cancer therapy for early-stage hormone-receptor positive (HR+) breast cancers (BCa). The selective estrogen receptor modulator (SERM), Tamoxifen (Tam) is effective for pre−/peri-menopausal women while post-menopausal women receive anti-estrogen aromatase inhibitors (AI). However, intrinsic or acquired ET-resistance is common and equates to rapid onset of malignant metastatic disease. Hence, HR+ BCa unresponsive to conventional ET poses a new challenge with no targeted therapy available to prevent cancer progression.The androgen receptor (AR) is emerging as a good therapeutic target for other advanced BCa subtypes . CanonicThe AR is subject to several post-translational modifications (PTM) including Small Ubiquitin Like Modifier (SUMO)-PTM , 11. ProWe now report that high levels of SUMOylated AR persist independent of AR-ligand in ET-R HR+ BCa cells. In these BCa lines, SUMO-PTM supports constitutive AR genomic activity to promote metastatic transformation. Concurrent targeting of SUMOylated AR improves the inhibitory actions of Enz on the drug-resistant HR+ BCa.2. MCF-7-TamR cells were generated in our lab by chronically treating MCF-7 with 1 μM 4-OH-tamoxifen (Sigma-Aldrich) for over 6 months. After developing resistance, the derived cells were used and continuously cultivated in presence of tamoxifen. All transient transfections were performed with Lipofectamine 2000 (Invitrogen) according to manufacturer instructions.Sensitive (MCF-7) and resistant  human breast carcinoma cell lines to ET were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CON-ethylmaleimide (Sigma-Aldrich), proteins were either immunoprecipitated from whole-cells or chromatin fractions using the same protocol described before [After harvesting and lysing the cells in a buffer containing protease inhibitor (Roche) and d before . ImmunopWhole-cell or immunoprecipitated protein extracts were denatured, resolved on SDS-PAGE gels and transferred onto PVDF membranes (BioRad) using a wet transfer apparatus. After blocking, blots were probed overnight and the following primary antibodies were used: AR (sc-816), Ubiquitin (sc-8017) and GAPDH (sc-8017) from Santa Cruz Biotechnology, AR441 (MA5–13426) and Hsp27 (MA3–015) from ThermoFisher, SUMO1 , SUMO2/3 , PIAS1 , and β-actin (Sigma-Aldrich). Antigen–antibody complexes were then detected by the chemiluminescence Western Lighting Plus-ECL reagent (PerkinElmer).t-test or one-way ANOVA followed by Tukey’s multiple comparison test was employed to evaluate statistical significance between groups and p-values < 0.05 were considered statistically significant.All results are expressed as mean ± SEM. Prism 7 (GraphPad Software) was used for data analysis. Student’s two-tailed Remaining methods used in this study are found in Additional file p = 0.0076, HR = 0.56 versus p = 0.18, HR = 2.21, respectively Fig. Elevated RNA levels of AR were previously reported in metastatic TamR BCa tumors . We analConsistent with a previous report , both lop < 0.05; Additional file p = 0.027; Additional file +-BCa patients with concurrent elevated levels of AR and SUMO exhibit a greater risk for developing metastasis . Also, induction of SUMO, analogous to native TamR conditions, enhances the interaction of AR with Hsp27, but not PIAS1  is readily detected and shifts from the cytoplasm to the nucleus with the overexpression of SUMO3 in TamS MCF-7 cells Fig. a. A sign1/2 > 24 h in TamR versus T1/2 ≈ 13 h in TamS  as compared to empty vector (T1/2 ≈ 8.2 h). However, SUMO overexpression produced no significant difference in the degradation rate of upper band modified AR , AR antagonist (Enz), or a combination of GA and Enz (GA + Enz). Concurrent GA + Enz treatment significantly inhibited migration and scratch gap closure of TamR-7 at all time points as compared to control or either drug alone Fig. a-b. We aWe and others observe that TamR-derived MCF-7 BCa cells grow efficiently in non-adherent spheroid cultures than parental MCF-7 cells consistent with their more metastatic phenotype , 32. HenIn this study, we report enrichment of SUMO-modified AR with ligand-independent activity in ET-R BCa as supported with the following results. First, while the unmodified AR is present in hormone-sensitive MCF-7 cells, the higher molecular weight SUMO-AR accumulates in cells with acquired (TamR-7) and intrinsic (GI101A and GILM2) resistance to tamoxifen endocrine therapy, and long-term estrogen deprived cells Fig. c. Third,Clearly tamoxifen therapy impacts how AR directs disease progression; unlike the general HR+ BCa patient population, high AR in patients receiving tamoxifen favors a worse prognosis Fig. a-b. In tFor AR, induction of SUMO is not sufficient for AR SUMOylation. In our cell-free system, we demonstrate that the SUMO-E3 ligase Hsp27 is required for SUMO-PTM of AR (Fig. AR agonist DHT stimulates transcription of mesenchymal genes and metastasis of MCF7 cells in xenograft studies . In MCF7Collectively and as illustrated in Fig. Our data suggest that dual targeting of AR and SUMO might have clinical and therapeutic relevance in the ET-R HR+ BCa subset of breast cancer. Therefore, increased global SUMOylation and in particular SUMO-modified AR may be a potential predictive biomarker useful to stratify HR+ BCa patients that are most likely to benefit from combining AR antagonists with SUMO inhibitors.Additional file 1. Supplemental materials and methods: in vitro SUMOylation; RT-PCR; Luciferase reporter assays; PLA, mammosphere studies; scratch assays; transcriptomic data analysis. Table S1. List of primer sequences used for the detection of transcripts. Supplemental figures and figure legends: Figure S1. Elevated levels of SUMO isoforms and HSP27 correlate with high probability of metastasis in ET-treated HR+ BCa patients. Figure S2. HyperSUMO conditions promotes AR SUMOylation and enhances its interaction with Hsp27. Figure S3. SUMO stabilizes AR and reduces its proteasomal degradation. Figure S4. SUMO stimulates basal AR transcriptional activity regardless of the AR-luciferase reporter construct. Figure S5. Concurrent targeting of SUMO-modified and unmodified AR decreases TamR-7 BCa growth in 3D cultures.


The transcription factor foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. We profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. We also observed changes to the axonal cytoskeleton and an accumulation of late endosomes in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, we identify several downstream factors mediated through foxo-regulated NMJ homeostasis, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. Our work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling. Aging involves the progressive functional decline of cellular mechanisms and tissue integrity Rose, . In the Drosophila NMJ synapse utilizes glutamate as the primary neurotransmitter and has postsynaptic densities that behave similarly to the AMPA-type receptors in the vertebrate central nervous system  promotes cytoskeletal dynamics at the neuromuscular junction during larval development, where it responds to stimulation to allow rapid structural reorganization. In the Drosophila larvae NMJ, loss of foxo results in increased stability of microtubules through the upregulation of kinesin motor protein regulator Paverotti that also controls axon growth   . We alsobranches . Thus, tC. elegans, late endosome formation is suppressed in organisms with delayed aging  at the synapse, which can result in neurodegeneration   . Interes < 0.05) .Ok6-gal4 driver and UAS-foxo-RNAi. Through the screening, we identified four genes whose knockdown attenuated the accumulation of late endosome number in foxo KD flies. We also found that overexpression of two genes Thor and Ank2 significantly reduced Rab7 number of the foxo KD flies. Among identified target genes, three genes, JNK transcription factor kay (Kayak), type I activin receptor babo (baboon), and p38b MAP kinase (p38b), are known to have regulatory effects on axonal transport , which is the Drosophila ortholog of ERK1/2 , ywR;+:foxo21, w1118, foxoC431 , Ok6Gal4 , mhcGal4  were crossed to double balancer yw;Sp/Cyo;TM2e/TM6BTbHue (Rochele). Markers were used to select carriers. After Ok6 was combined with the foxo-RNAi, lines were backcrossed for 5 generations before use. Flies containing Ok6 in a yw background were self-crossed to produce a control strain. Line was backcrossed for 5 generations. A daughterless gene switch GAL4 driver was used to validate foxo knockdown and overexpression.The following stocks were used: 2+ free saline . Tissue was then fixed in 4% paraformaldehyde for 20 min at room temperature. Tissue was washed in 1X PBST (0.1% Triton X) and blocked with 5% NGS  for 1 h at room temperature. Samples we incubated overnight at 4°C with 2 ug primary antibodies in 5% NGS 1X PBST . Primary antibodies used were as follows: anti-BRP (DSHB nc82), anti-Dlg (DSHB 4F3), anti-Rab7 (DSHB), anti-Rab7 (gift from Nakamura lab), anti-Acetlyated Tubulin (Sigma Aldrich T7451), anti-phospho-p38 MAPK (CST 9211). Tissues we washed in 1XPBST and incubated 1.5 h at room temperature in 1XPBST and anti-HRP-594 or HRP-647  at a Dilution of 1:200 with secondary antibodies (1:250) and kept in the dark at room temperature for 2 h. Samples were washed in PBST and mounted in Prolong Diamond (Life technologies). For Fat body analysis, pelts were incubated in slowfade gold with DAPI (life technologies) at 4°C overnight prior to mounting.Flies were subjected to flynap  and dissected to expose the ventral abdominal muscles [protocol from Wagner et al. ] in Ca2+All secondary antibodies used were from Jackson ImmunoResearch Laboratories Inc and were as follows: 488 donkey anti-mouse, 488 goat anti-rabbit, rabbit anti-HRP-594, donkey anti-HRP-647.2) to quantify number of punctae. Z-slices quantified were limited to those containing HRP staining at selected regions to avoid non-neuronal associated punctae. ROIs were checked prior to quantification to ensure muscle associated punctae were removed. Intensities for pp38, pERK, and pJNK were conducted using Fiji (NIH) by measuring the sum fluorescence intensity in ROIs defined by HRP immunoreactivity. HRP fluorescence intensity was measured to ensure no differences between control and mutant NMJs. All images were generated using Fiji.Images were captured using an epifluorescence-equipped BX51WI microscope  and Olympus FV3000 laser scanning confocal . CellSens software  was used for deconvolution of stacks. For aging metrics quantifications, maximum Z projections were generated of whole A3 muscle segment. Bouton size and active zone number were quantified in CellSense through a selected region of interest (ROI) aligned to each bouton. Measurements were performed using the “measure and count” module, providing quantification of bouton and active zone number and area. Branch number and branch length were quantified in Fiji , Rpl32 .Three-day post eclosion flies were placed on food containing < 0.1% ethanol and 200 uM Mifepristone  or no other additive for 3 days. Whole bodies of female flies were homogenized in TRIzol (Thermo Fisher). RNA was extracted followi ng company procedure. cDNA was generated using qScript (Quanta) and diluted to a 10 uM working solution. Quantitative PCR was run on purified samples (QuantStudio). Sybrrgreen  was used for chemical detection. The primers used Enrichment was determined based on the double-delta CT value. Stacks of the entire A3 muscle region were acquired with Olympus FV3000 laser scanning confocal in the Fluoview software . Videos were recorded of 3D renderings using the movie module under the volume setting.http://bioinformatics.psb.ugent.be/webtools/Venn/.Venn comparisons were performed using the Bioinformatics and Evolutionary Genomics Venn calculator at t-test or one-way ANOVA was performed using Tukey multiple comparison. All genetic screen lines were compared to the generated foxo-RNAi line using a student t-test. The metrics during aging were analyzed by two-way ANOVA, including Tukey multiple comparisons test. Before analysis, outliers were identified using Robust regression and Outlier removal (ROUT) method (Q = 1%).GraphPad Prism (GraphPad Software) was used for statistical analysis. To compare the mean value of treatment groups vs. that of control, either student The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AB, MS, MB, KC, and PK: data acquisition. AB, EM, and HB: design, analysis, interpretation, and manuscript revision. AB and HB: conception. AB: manuscript writing. HB: resources and funding acquisition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TN) gradually decreases from 70 to 45 K as the thickness declines from 32 to 5 nm. And ferromagnetic hexagonal FeTe is accompanied by a drop of the Curie temperature (TC) from 220 K (30 nm) to 170 K (4 nm). Theoretical calculations indicate that the ferromagnetic order in hexagonal FeTe is originated from its concomitant lattice distortion and Stoner instability. This study highlights its potential applications in future spintronic devices.Two-dimensional (2D) magnets with intrinsic ferromagnetic/antiferromagnetic (FM/AFM) ordering are highly desirable for future spintronic devices. However, the direct growth of their crystals is in its infancy. Here we report a chemical vapor deposition approach to controllably grow layered tetragonal and non-layered hexagonal FeTe nanoplates with their thicknesses down to 3.6 and 2.8 nm, respectively. Moreover, transport measurements reveal these obtained FeTe nanoflakes show a thickness-dependent magnetic transition. Antiferromagnetic tetragonal FeTe with the Néel temperature ( Two-dimensional magnets with intrinsic ferromagnetic/antiferromagnetic ordering are highly desirable for future spintronic devices. Here, the authors demonstrate a chemical vapor deposition approach to controllably grow ultrathin FeTe crystals with antiferromagnetic tetragonal and ferromagnetic hexagonal phase, showing a thickness-dependent magnetic transition. The discovery of long-range magnetic order in 2D van der Waals (vdW) crystals is vital to the understanding of the spin behavior in 2D limit and can enable extensive spintronic applications ranging from molecular quantum devices to high-density data storage devices5. Different from conventional magnetic thin films and their bulk counterparts, these intrinsic 2D magnetic crystals show very peculiar properties and often bring about different physical phenomena. For instance, layer dependent ferromagnetism with an out-of-plane anisotropy was observed in CrI3 and Cr2Ge2Te6 nanosheets at low temperatures6. Furthermore, ferromagnetic ordering with Curie transition temperature (Tc) > 300 K was induced in Fe3GeTe2 extrinsically by an ionic gating method7. In addition, long-distance magnon transport was also detected in the antiferromagnetic MnPS3 crystal8.Recently, 2D magnets have drawn much attention because they provide an ideal platform for exploring magnetism down to atomic-layer thicknesses3). Therefore, either glove box-involved mechanical exfoliation or molecular beam epitaxial (MBE) method has to be employed to prepare the 2D magnets in an oxygen/water-free or ultra-high vacuum condition. Also, these approaches are time-consuming and not industrial scalable10. Second, many 2D magnets show intricate phases . Hence, it is challenging to identify an exact phase of these 2D magnets and provide a sound explanation for the origin of its magnetism. Recently, selective substrates, such as mica, were chosen for the growth of a few of 2D magnets, including ultrathin Cr2S3 and CrSe crystals12. Such epitaxial growth technique can only be applied to a limited number of 2D magnets and it also raises a transfer problem during the device fabrication or magnetic measurements. For instance, the transfer process inevitably introduces defects and contamination, which dramatically degrades the performance of devices. Thus, exploring more 2D magnetic compounds and developing reliable synthesis methods are urgent and necessary.However, two issues hinder the practical applications of 2D magnets. First, some 2D magnets are extremely unstable , a variety of magnetic behaviors, such as ferromagnetism, ferrimagnetism, and antiferromagnetism can be observed16. In addition, Fe-chalcogenides also exhibit multiple structural phases with distinct properties18. For instance, the conventional phase of FeTe is an antiferromagnetic metal with a tetragonal crystal structure and TN ≈ 70 K16. Recently, density-functional theory (DFT) calculations predicted an intriguing hexagonal FeTe phase, which is weakly magnetic19. As the magnetic properties of FeTe can be significantly affected by the changes in structural phases, controlled synthesis of FeTe with tunable structural phases is needed. So far, the synthesis of 2D FeTe crystals by CVD method has been rarely reported, much less the phase control20.Fe-chalcogenides  are a recent class of magnetic materials2/Si substrates. By controlling the growth temperature, ultrathin 2D-layered tetragonal FeTe nanoplates and non-layered hexagonal FeTe nanoplates were selectively obtained as square or triangular shapes, with thicknesses down to 2.8 and 3.6 nm, respectively. The grain size of FeTe flakes is up to dozens of micrometers, suggesting they are high-quality single crystals. Furthermore, the antiferromagnetic behavior of tetragonal FeTe is exhibited with a TN decreases as the thickness decline, while the ferromagnetic hexagonal FeTe is also displayed with a thickness-dependent TC. Theoretical calculations indicate that the structural distortion is responsible for the observed ferromagnetism.In this work, we have directly synthesized antiferromagnetic and ferromagnetic FeTe on SiO2/Si substrates. Briefly, ferrous chloride (FeCl2) and tellurium (Te) powders were used as reactants. During the CVD process, the Ar and H2 mixture carries a certain amount of tellurium vapor at a fixed heating temperature of tellurium source (TTe) to react with evaporated FeCl2 at different growth temperatures (Tgrowth) to produce different phases. Details about the sample synthesis are provided in the Methods section. As shown in Supplementary Fig. P4/nmm space group owning a layered structure in which Fe atom layer and the double slabs of Te atom layers are interlaced in the interlayer direction . As shown in Fig. Tgrowth will lead to the formation of tetragonal FeTe. A clear trend can be seen from the following optical images  image in Fig. tal Fig. . Supplemtal Fig.  and 5 deZ atomic number varying approximately as Z1.6–1.7, and thereby the Z-contrast STEM image is widely employed to identify the atomic structures in 2D materials28. The FeTe crystals were transferred from the SiO2/Si substrate to TEM grids, as shown in Supplementary Fig. 30. The transfer details are provided in the “Methods” section. A typical atomic-resolution STEM-ADF image of the tetragonal shaped FeTe crystal along the [001] zone axis was depicted in Fig. 4g wallpaper group symmetry. The in-plane Te–Te and Fe–Fe bonds are estimated to be ~3.0 Å, as verified by the zoom-in STEM image  imaging was applied. The image contrast in STEM-ADF image is intimately tied to the age Fig. . No discage Fig. . Apparenage Fig.  is homogIn stark contrast to the tetragonal shaped FeTe crystal, the STEM image Fig.  of hexag12. For instance, bulk-sensitive techniques inevitably detect the signal from magnetic contaminations, because ultrathin samples’ weak magnetic signal is often comparable or even weaker than that of contaminations obtained from various sources, such as substrate, glue, magnetic particles from laboratory environment. Supplementary Fig. 2/Si substrates and well-protected by the hexagonal boron nitride (h-BN) capping layer. We should note that tellurides are susceptible to ambient degradation. Generally, our FeTe crystals can survive in the air for only half an hour  and superconducting quantum interference device (SQUID), magnetoelectric measurement is more suitable to characterize the weak magnetism from low dimensional nanoflakesTc ~ 220 K for 30 nm, Tc ~ 215 K for 12 nm and Tc ~ 170 K for 5 nm, respectively. The ferromagnetism was further confirmed by the Hall measurement. As for the thick hexagonal FeTe (30 nm), the anomalous Hall effect (AHE) observed clearly at 1.5 K within ±0.5 T magnetic field range  gradually decrease, above which the AHE vanishes  and FM–AFM  configurations, which are yet to be confirmed by low-temperature STEM experiments. Details of all considered magnetic orders are available in Supplementary Fig. c shows a considerable shrink from roughly 5.8 to 5.45 Å  and an increased angle α (from 120.0° to 121.4°)  Fig. . The broTc value around 290 K using Metropolis Monte Carlo (MC) simulation in a 3-layer 3D Ising lattice. Given the fact that MC simulation of the Ising model usually overestimates Tc two or three times32, the hexagonal phase is expected to have a Tc value around 100–150 K in consideration of the Heisenberg local exchange picture, substantially lower than the measured value of ~220 K. This low Tc value indicates another mechanism may corporately result in such a high Tc value of 220 K. Stoner ferromagnetism mechanism is another likely reason for the FM. Non-magnetic calculations were performed to calculate the DOSs using the FM relaxed geometries. Figure d-orbital DOSs of the undistorted and distorted structures, respectively, both of which meet the Stoner criterion, i.e., (U′/N)ρ(Edf) > 1, compellingly indicating the Stoner ferromagnetism in this metallic system. In addition, the distorted structure shows a larger (U′/N)ρ(Edf) value (1.95) than that of the undistorted structure (1.55), evidencing a stronger Stoner ferromagnetism in distorted FeTe  of the Te-terminated hexagonal slab in a Te-rich condition, roughly an order of magnitude smaller than those of other slabs. Thus, the Te-terminated hexagonal slab is the most energetically favored one among all hexagonal and tetragonal slabs in the Te-rich growth condition that could be reached by increasing the Te source temperature, as we demonstrated in our experiments. In addition, our vibrational frequency calculations indicate that Te-terminated hexagonal FeTe mono- and few-layers are, exceptionally, unstable and tend to transform into Fe–Te clusters at 0 K, with imaginary frequencies of e.g., 79.21 and 188.83 i cm−1 for the monolayer. Given the experimentally synthesized hexagonal phase, we infer that the few-layers hexagonal FeTe is most likely, stabilized by non-harmonic effects, which suppresses the phonon softening and is more pronounced at high temperatures33. This partially explains the reason why the hexagonal phase is a high temperature phase.Vibrational frequency calculations were performed to compare free energies of different phases. The free energy plot ), which had three independently controlled temperature zones. Te powder  and ferrous chloride  were used as solid precursors and reactants. The Te powder (in a quartz boat) was placed near to the gas inlet in the first heating zone while the ferrous chloride powder, loaded into the third heating zone, was placed in an alumina boat covered with an inclined downward-facing SiO2/Si substrate. The second heating zone was kept at the same condition as that of the first heating zone. Prior to the growth process, the furnace was purged by the flowing of 300 sccm high-purity Ar gas for 5 min. Then, 100 sccm Ar and 10 sccm H2 mixtures were introduced into the CVD system. The tellurium was heated to 520 °C in 20 min while the growth zone of ferrous chloride was gradually increased to different temperatures (520–630 °C) in 20 min. Set temperature for all the heating zones was kept for another 10 min for growth of temperature. After synthesis, the furnace was rapidly cooled down to room temperature with the assistance of electric fans. Obvious etching phenomena were observed when the furnace was naturally cooled without any external perturbations for both tetragonal and hexagonal FeTe flakes , wh2/Si substrate to TEM grids followed a surface-energy-assisted transfer approach30, which was shown in , which floated on the surface of 1 mol L−1 KOH solution due to the surface tension of water  to around 20 ppm. However, the entire process only lasts for a few minutes. And we kept the open of cleaning gear button for the glove box during the transfer process. Thus, we can work with aqueous solutions for a short time inside a glove box.The transfer of the FeTe flakes from SiO2/Si substrate were quickly moved to an Ar glove box and spin coated with 950 K PMMA (MicroChem) at 3000 r.p.m. for 1 min. After leaving the PMMA layer consolidating on a 120 °C hotplate for 5 min in the Ar glove box, small markers were fabricated using standard e-beam lithography ) near the identified sample for subsequent fabrication of Hall-bar devices. The Ti/Au (5/70 nm) electrodes were deposited using an electron-beam evaporator  followed by lift-off in acetone. The electrical contacts were made by wire bonding Al wires onto the six electrodes on a Hall-bar structure. Transport experiments were performed in an Oxford cryostat with the temperature ranging from 300 to 2 K and the magnetic field up to 8 T. The sample resistance and Hall effect were measured by Keithley 6221 triggered with Keithley 2182 with the 21 Hz frequency.The tetragonal and hexagonal FeTe flakes grown on SiOThe as-obtained FeTe nanoplatelets were further characterized by optical microscopy (Olympus BX53M), RM , AFM (Asylum Research Cypher Scanning Probe Microscope system with a tapping mode), and XPS . ADF-STEM imaging was conducted on an aberration-corrected JEOL ARM-200F equipped with a cold field emission gun, operating at 80 kV, and an Advanced STEM Corrector (ASCOR) probe corrector.35 as implemented in the Vienna ab-initio simulation package (VASP)37. The Perdew–Burke–Ernzerhof (PBE)38 functional with the density-dependent dispersion correction (dDsC)40 was adopted for all calculations. A uniform Monkhorst–Pack k-mesh of 21 × 21 × 13 was adopted for sampling over the Brillouin zone of hexagonal FeTe and a 20 × 20 × 8 mesh for the tetragonal phase. A plane-wave kinetic energy cutoff of 700 eV was used for structural relaxation while 400 eV for energy comparison. All atoms were allowed to relax until the residual force per atom was less than 0.01 eV Å−1. Full relaxation of all atomic coordinates using the dispersion-corrected PBE functional well addresses the Te-height-dependent magnetic ground state issue41, and finds the correct magnetic ground state for tetragonal FeTe16. We also consider spin-orbit coupling (SOC) for energy comparisons among different magnetic configurations. A 2 × 2√3 × 1 orthorhombic supercell, with a k-mesh of 4 × 4 × 6, was used to model different magnetic orders in FeTe. An on-site Coulomb interaction energy to Fe d orbitals were self-consistently calculated using a linear response method42, which revealed U = 3.3 eV and J = 0.5 eV.Our density-functional theory calculations were performed using the generalized gradient approximation and the projector augmented wave methodJ1 and J2 and three interlayer ones, J3 to J5, respectively, as displayed in Supplementary Fig. Tc value in a three-layer lattice using a 3D Ising model, S = 2. A 20 × 20 × 3 lattice was used with a periodic boundary condition.A Heisenberg model was used to model the magnetism of the bulk FeTe crystal, mentclasspt{minimaa = 3.90 Å and b = 3.82 Å to calculate the DOS of the distorted structure. We speculate that no structural distortion is acquired at room temperature, since both geometric relaxation and STEM show no significant distortion. What we show in (b) only tends to discuss how distortion affects the DOS. Stoner parameter is defined as ST = (U′/N)ρ(Edf). Here, N is the number of unit magnetism cells, which equals to 2 in this situation and ρ(Edf) is the total density of states of all d orbitals at the Fermi level for hexagonal FeTe . ΓMKZAR in the BZ, which could be derived from the band structure plotted in Supplementary Fig. md is the average value of the magnetic moments on Fe atoms, i.e., 3.4 and 2.9 μB for undistorted and distorted FeTe.Structural relaxation of non-magnetic configuration does not show structural distortion. We thus kept the in-plane constants Supplementary Information


Salt stress causes rapid accumulation of nonexpressor of pathogenesis-related genes 1 (NPR1) protein, known as the redox-sensitive transcription coactivator, which in turn elicits many adaptive responses. The NPR1 protein transiently accumulates in chloroplast stroma under salt stress, which attenuates stress-triggered down-regulation of photosynthetic capability. We observed that oligomeric NPR1 in chloroplasts and cytoplasm had chaperone activity, whereas monomeric NPR1 in the nucleus did not. Additionally, NPR1 overexpression resulted in reinforcement of morning-phased and evening-phased circadian clock. NPR1 overexpression also enhanced antioxidant activity and reduced stress-induced reactive oxygen species (ROS) generation at early stage, followed with transcription levels for ROS detoxification. These results suggest a functional switch from a molecular chaperone to a transcriptional coactivator, which is dependent on subcellular localization. Our findings imply that dual localization of NPR1 is related to proteostasis and redox homeostasis in chloroplasts for emergency restoration as well as transcriptional coactivator in the nucleus for adaptation to stress. Even though ROS play an important role as signaling molecules and inducers in the adaption of plants to abiotic stress, they are also toxic byproducts of stress metabolism4. Chloroplasts act as sensors of the present environmental situation5 and produce diverse signals communicating the functionality of the photosynthetic apparatus to the nucleus, which is defined as retrograde signaling6.Chloroplasts are particularly vulnerable to environmental disturbances, because of oxygenic photosynthesis8. Because the rapid generation of ROS represents a common plant response to almost all environmental challenges9, it is suggested that ROS and the redox system in chloroplasts represent primary sources within the plant signaling battery. This hypothesis implies there are interactions between ROS and other signaling components4 such as redox homeostasis, plant hormones, and transcription factors4.Recent genomic technologies provide growing evidence that ROS generation is one of the most common responses to different stresses in plants, representing various signaling pathways come together10. Nuclear-encoded transcripts for chloroplast proteins may be related to the circadian regulation of chloroplasts11.Sunlight for photosynthesis is available only for a limited period within the 24 h day. The rhythmic and predictable alteration of solar energy has driven the evolution of the circadian clock, which is integrated with signals within chloroplasts12. In addition, ROS or oxidized small molecules are involved in aberrant modifications of protein structure, which eventually leads to toxic effects on cells13. Therefore, chloroplasts have well developed mechanisms protecting against protein modifications via molecular chaperones that bind reversibly to unfolded and misfolded proteins, thereby maintaining native protein conformation. Therefore, chaperones are now considered as powerful buffers for multiple stress resistance14.Proper protein folding and localization are critical for cellular protein function. However, cells are exposed to environmental stresses, which makes them susceptive to nonnative condition that ultimately can result in misfolding and aggregation17. In unstressed cells, NPR1 is mostly localized in the cytoplasm as a tetrameric complex with redox-sensitive intermolecular disulfide bonds15. Pathogen infection triggers alteration of cellular reduction potential, thereby reducing NPR1 tetramer into a monomer via breakage of disulfide bonds, after which NPR1 monomer is imported into the nucleus to function as a coactivator of gene transcription18. Nuclear NPR1 interacts with transcription factor (TF) members of the TGA class of basic domain/leucine zipper19. However, there are exceptional cases in which nuclear localization of NPR1 is not a requirement for SA-dependent gene regulation20, indicating SA-activated cytosolic NPR1 has a novel function beyond transcriptional coactivator in nucleus18.Numerous studies have revealed that nonexpressor of pathogenesis-related genes 1 (NPR1) protein is a master regulator of plant immunity with salicylic acid (SA)-mediated defense responses and systemic acquired resistance (SAR) in ArabidopsisNicotiana tabacum and Arabidopsis thaliana under salt stress and SA treatment. NPR1 in the chloroplasts was shown to have the function of chaperone activity and redox regulation as well as transcriptional coactivator in the nucleus in response to salt stress, suggesting NPR1 might be involved in proteostasis and redox homeostasis. The novel features of chloroplasts related with NPR1 are very effective in promoting adaptation against abiotic/biotic stress and developmental regulation in plants.Here, we observed that NPR1, a so-called nucleocytoplasmic protein, transiently accumulated in the chloroplast stroma of 21, NPR1 has not been fully characterized in relation to abiotic stresses. Here, we observed that the transcription levels of NPR1 rapidly increased from 1 h to 6 h, after which they gradually returned to basal levels in leaves of Nicotiana tabacum after salt stress 22. NPR1-Ox plants showed greater tolerance to salt stress, as determined by trypan blue staining for cell death22 23 based on chlorophyll fluorescence using a PAM 2000 Photosynthesis Yield Analyzer  . However, the ratio of Fv/Fm was reduced only by 24% after 96 h of salt stress in NPR1-Ox leaves.Although it is well known that NPR1 protein is a key transcriptional coactivator in broad-spectrum immunity of plants against phytopathogensess Fig. . To inveh22 Fig.  as well ny) Fig. . Under s24. Stress-triggered transcriptional down-regulation of chloroplast-encoded large subunits (RbcL) and nucleus-encoded small subunits (RbcS) was mitigated in NPR1-Ox compared to WT  was performed with ribulose-1,5-bisphosphate carboxylase (RubisCO) genes responsible for photosynthesis WT Fig. . RbcL trNPR1-Ox upon salt stress. Real-time qRT-PCR was performed using genes for RubisCO and core complex and antenna proteins of PS I and II. The transcriptional ratios of NPR1-Ox to WT were obtained on the basis of each relative expression value for chloroplast- and nucleus-encoded genes using β-actin as the reference gene. Transcriptional ratios of chloroplast- and nucleus-encoded genes above 1 indicated higher gene expression in NPR1-Ox compared with WT  and CIRCADIAN CLOCK ASSOCIATED (CCA1) and the evening clock gene of TIMING OF CAB2 EXPRESSION 1 (TOC1)29.The production of sugars by photosynthesis is the main metabolic outcome of circadian clock in plantsLHY and TOC1 was detected in NPR1-Ox than WT, and the transcription levels of both genes were significantly increased in salt stressed-tobacco plants relative to mock treated-tobacco plants  (p35S::AtNPR1-GFP). It is well known that NPR1 is a nucleocytoplasmic protein in Arabidopsis15. However, after transient expression in mesophyll protoplasts from Arabidopsis leaves, AtNPR1-GFP was astonishingly detected in chloroplasts under salt stress 22. NPR1-GFP accumulated in chloroplasts of mesophyll protoplasts from this transgenic tobacco plant -1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), are known to inhibit the electron transport chain in PS II33, showed a similar level of chloroplast NPR1 accumulation with salt treatment in leaf protoplasts , a compound inducing strong photo-oxidation and subsequent plastid dysfunctionsts Fig. , suggeststs Fig. . These r35, was administered to transgenic plants of NPR1-GFP. As treatment with Lin began, the fluorescence intensity of stress-induced NPR1 decreased significantly in chloroplasts , a translation initiation inhibitor in intact chloroplastssts Fig. . These r36. A synergistic effect between ROS and ethylene production in tobacco plants was previously reported37. Different members of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes were involved in the biphasic phase of ethylene production: NtACS4 in the early phase at 1–3 h and NtACS1 in the late phase at 48–72 h after pathogen infection38. Therefore, we used NtACS4- and NtACS1-silenced transgenic plants (NtACS4i and NtACS1i) via RNA interference-mediated repression. Stress-induced NPR1 localization in chloroplasts was significantly reduced in transient expression of pNPR1::NPR1-GFP with antisense transgenic plants of NADPH oxidase genes (RbohD and RbohF) and RNAi expression of ethylene biosynthetic genes (NtACS4i and NtACS1i)  Fig. . Our resTo unravel the dependence of chloroplast NPR1 accumulation on other plant hormones, we analyzed the spatial pattern of NPR1-GFP protein in leaves after treatment with several hormones. Interestingly, fluorescent NPR1 significantly accumulated in chloroplasts in response to all tested plant hormones: indole-3-acetic acid, 6-benzyladenine, gibberellin, SA, jasmonic acid (JA), and ACC as an ethylene precursor, and ABA  antibody, no contamination with the chloroplast outer envelope was detected Fig. . Furtherp35S::AtNPR1-GFP transient expression in mesophyll Arabidopsis protoplasts  were detected in chloroplast stroma proteins after sts Fig.  and in pves Fig. . The olives Fig. .pNPR1::NPR1-GFP transgenic tobacco plants at 6 h upon salt stress, and these molecules were named CP45 , is nucleus-encoded, and thus enters internal chloroplast compartmentsnce Fig. . The posnce Fig. , whereas14. Therefore, NPR1, as a transcriptional partner, stimulates the DNA-binding activities of TGA34 and induces pathogenesis-related (PR) gene expression in plant immune responses42. We next investigated the efficacy of nuclear-localized NPR1 as a transcriptional activator. Transcription of TGA2, an NPR1-dependent tobacco orthologue of TGA transcription factor1543, was effectively induced in response to salt stress and peaked at 12 h in NPR1-Ox  chaperone is an essential regulator of proteostasis in eukaryotic cells WT Fig. . However48 and mitochondria of eukaryotic cells49. Chloroplast-encoded ClpP is a core subunit of Clp protease, which is the most abundant stromal protease in chloroplasts48. In the unstressed control, the transcription level of ClpP was 2-fold higher in NPR1-Ox transgenic compared with WT leaves, indicating NPR1 contributes to plastid protein homeostasis -So and BES-H2O2, respectively22. Stress-induced ROS levels were significantly inhibited in chloroplasts of NPR1-Ox compared to WT, suggesting that ROS generation might be closely related to the localization of NPR1 in chloroplasts , catalase, and glutathione-S-transferase phi51. It has been recently reported that concentration-dependent regulation by ROS is essential in signaling pathways for stress resistance52, and thus the concentration levels of ROS are a determinant of cell viability52. The dual nature of ROS, either beneficial or detrimental, underscores the need to identify thresholds that determine which direction will act in the cells52. In this study, we observed that plant cells under salt stress rapidly up-regulated a redox-sensitive NPR1 protein, which translocated to chloroplasts for induction of protective responses while lowering chloroplastic ROS accumulation 22. In WT leaves, oxidized proteins in chloroplasts accumulated immediately from 2 min after salt stress, reaching a peak at 5 min and then maintaining a high level until 3 h 57, including not only transcription factor-binding and protein dimerization activity but also binding to unfolding proteins and chaperone. The main reason why NPR1 has been shown to represent these functions in the InterPro database is that NPR1 has several Ankyrin repeats, which are degenerate 33–amino acid repeats that serve as domains for protein–protein interactions58. Proteins with ankyrin repeats have been reported to act as chaperones. Therefore, it is possible that chaperone activity of NPR1 was mediated by binding to a chaperone or by direct chaperone function.The hydrophobic structure of NPR1 may contribute to protein stability and polymeric form in chloroplasts, and its function was shown to be related with chaperone activity in a stress-dependent manner Fig. . NPR1 wa46. Proteostasis mediated through the chaperone activity of chloroplast NPR1 was shown to be related to stress tolerance of enhanced chloroplast gene expression, cell viability, and photosynthetic capability with maximal quantum yield of PS II , after which it enters a secondary compartment (nucleus). Our studies have demonstrated this possibility and NPR1 should be further studied since it has been shown to participate in signaling movement as well as have dual functions in chloroplasts and the nucleus.More interestingly, our results imply that NPR1 has different functions in chloroplasts and the nucleus. The switch of NPR1 function is related to the intracellular redox status originating from environmental conditions, indicating that NPR1 can serve as a communicator and provide emergency restoration after sensing stress-induced redox status. The original dogma that every polypeptide fulfils only one function has been replaced by the notion that many are bi- or even multifunctionalNicotiana tabacum L. Wisconsin 38) plants were grown on solid Murashige and Skoog (MS) medium (pH 5.8) under light  at room temperature (25 ± 5 °C), which were followed by the previously reported experimental method17. Fully-matured WT and T1, T2, or T3 plants after antibiotic selection were subjected to salt stress (200 mM NaCl) or salicylic acid (SA) treatment (300 μM). Solutions with salt, SA, and other chemicals were applied to whole leaves with petiole in 20 mM MES buffer under light (100 μM photons m−1s−1) at 25 °C. For mock treatment, tobacco leaves or whole plants were floated on MES buffer without any chemicals17.Surface-sterilized seeds of tobacco  of AtNPR1 (GenBank: ATU76707) was PCR-amplified and the resulting product cloned into pMBP vector harboring 35 S promoter-driven green fluorescence protein (GFP) gene and NOS terminator. For p35S::NPR1-GFP, the open reading frame (ORF) of NPR1 (GenBank: KY402167) was PCR-amplified and the resulting product cloned into pMBP vector harboring 35 S promoter-driven green fluorescence protein (GFP) gene and NOS terminator. In control transgenic plants with free GFP, pMBP vector with 35 S promoter-driven GFP gene alone and NOS terminator was used as a transgenic construct. PCR-amplified full-length of NPR1 was overexpressed by cloning into pMBP vector harboring 35S promoter and NOS terminator after removal of the GFP gene, which was named NPR1-Ox construct. Further, native NPR1 promoter from genomic DNA of Nicotiana tabacum was amplified by PCR, after which the DNA fragment was gel-purified using a QIA quick Extraction Kit (Quigen). The 0.8 kbp DNA fragment of NPR1 promoter was cloned into promoter-less NPR1-GFP construct, which was prepared from p35S::NPR1-GFP after deletion of 35S promoter fragment. Resulting constructs were introduced into N. tabacum by Agrobacterium (strain LBA 4404)-mediated transformation. Successfully transformed T1 plants were kanamycin-resistant and then selected for further propagation. Lines with T2 progeny segregating 3:1 for kanamycin resistance:sensitivity were further propagated, and homozygous T3 plants were used for further study in the cases of NPR1-Ox and p35S::NPR1-GFP. In the experiments with pNPR1::NPR1-GFP transgenic plants, T1 plants were used after selection of kanamycin resistance. Surface-sterilized transgenic seeds were cultured on solid Murashige and Skoog medium (pH 5.8) under light  at room temperature (25 ± 5 °C)22.For 17. To analyze transcription levels by real-time qPCR, 1 μg of total RNA from leaves was reverse-transcribed for 30 min at 42 °C in a 20 μl of reaction volume using a High Fidelity PrimeScriptTM RT-PCR kit  according to the manufacturer’s instructions. Gene-specific PCR primers for qPCR were designed according to a stringent set of criteria . A 20 μl of reactions consisted of 10 μl of 2X SYBR Green Master Mix, 0.5 μM of each primer, and 10 ng of cDNA. PCR conditions were as follows: 95 °C for 15 min, 45 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, extended by 72 °C for 10 min. Fluorescence threshold data (Ct) were analyzed using Thermal Cycler Dice® Real Time System III Software version 6.0  and then exported to Microsoft Excel for further analysis. Relative transcription levels in each cDNA sample were normalized to the reference gene β-actin after qPCR. The mean relative mRNA expression level for each gene in WT and overexpressing transgenic plants (NPR1-Ox) was obtained, and the expression ratio for each gene between WT and Ox plants was calculated17.The isolation of Total RNA was performed as previously describedRbc S, RubisCO Small subunit; PsaF, Photosystem I reaction center subunit III; PsaK, Photosystem I subunit X; PsaN, Photosystem I reaction center subunit XII; CAB, Chlorophyll a/b-binding protein; CAB3, Chlorophyll a/b-binding protein 3; CAB21, Chlorophyll a/b-binding protein 21; HSP90, heat shock protein 90; CAB36, Chlorophyll a/b-binding protein 36; LHCB6, Photosystem II light-harvesting chlorophyll-binding protein CP24.Nucleus-encoded genes: Rbc L, RubisCO Large subunit; PsbA, Photosystem II reaction center D1 protein; PsbN, Photosystem II subunit; PsaB, Photosystem I reaction center; ClpP, Caseinolytic protease subunit P; PsaJ, Photosystem I subunit IX.Chloroplast-encoded genes: 59. To determine plant cell death, salt-treated tobacco leaves were immersed for 1 min in a boiling solution consisting of 10 ml of lactic acid, 10 ml of glycerol, 10 g of phenol, and 0.4% (w/v) trypan blue. After plants had cooled to room temperature, solution was replaced with 70% (w/v) chloral hydrate. Stained plants were decolorized overnight to removing chlorophyll and then imaged using a digital camera.This experiment was performed as previously described37. Steady-state net photosynthesis of eight-week-old whole plants was determined using a Gas Exchange Measuring Station  with a built-in light source (210 μmol photons m−2 s−1). Gas stream  was provided continuously into the measuring chamber by a mass-flow control system. Leaves were maintained at 25 °C and 70 ± 1% humidity conditions.Photosynthetic activity was measured as previously described35 S::GFP were used. Protoplasts were prepared by incubation in enzyme solution 22. GFP measurements were performed as previously described22. GFP fluorescence in cells was detected using a confocal laser scanning microscope  or a fluorescence microscope  equipped with a high resolution CCD camera. GFP expression was visualized by excitation at 488 nm and emission at 520 nm. Chlorophyll fluorescence was visualized by excitation at 458 nm and emission at 647–720 nm. Fluorescence of DAPI  staining for nuclei was visualized by excitation at 358 nm and emission at 461 nm. Fluorescence density was quantified with ImageJ bundle software provided by the Wright Cell Image facility.Transgenic expression of NPR1-GFP in leaves, roots, and protoplasts prepared from transgenic NPR1-GFP tobacco plants. For free GFP expression, control transgenic plants with 2O2  are fluorescent probes for superoxide and hydrogen peroxide, respectively59. BES-H2O2 was used at a concentration of 50 mM in 20 mM potassium phosphate buffer (pH 6) for 1 h in the dark . BES-So was used at a concentration of 20 mM potassium phosphate buffer (pH 6) for 1 h in the dark . Fluorescence was observed using a confocal laser scanning microscope FluoView 300 .For fluorescent detection of ROS, leaf epidermal strips were used. Leaf epidermal strips were peeled from tobacco leaves with salt stress after the indicated time. Leaf epidermal strips were floated on the indicated solution. BES-So and BES-H20.To extract total protein from tobacco leaves, frozen samples were ground into powder and suspended in protein extraction buffer  containing 50 μg/ml of tosyl-L-phenylalaninyl-chloromethylketone, 50 μg/ml of tosyl-L-lysine-chloromethylketone, serine protease inhibitors, 0.6 mM phenylmethylsulfonyl fluoride (PMSF), 80 μM MG115, 80 μM MG132, and complete protease inhibitor cocktail tablet 2, 1 mM NaF, 1 mM EDTA, 1 mM PMSF, 80 μM MG115, 80 μM MG132, 10 μM pepstatin A and one complete protease inhibitor cocktail tablet ). After lysate centrifugation, supernatants were recovered as total proteins or chloroplast stroma proteins. Inhibition of proteasome-dependent degradation was accomplished by 40 μM MG115.To isolate chloroplast stroma proteins from tobacco leaves, chloroplasts were first isolated from leaves using a chloroplast isolation kit , after which intact chloroplasts were harvested by 40/80% Percoll gradient. Intact chloroplasts were suspended in chloroplast lysis buffer . According the manufacturer’s protocol, nuclei were collected from leaves with nuclei isolation buffer by mesh filtering. Cell lysate was prepared with 2.3 M sucrose by centrifugation at 12,000 × g for 10 min, after which the supernatant was discarded. The nuclei pellet was then added to nuclear protein extraction buffer. Nuclei proteins were then added to Working Extraction Buffer in addition with 80 μM MG115 and 80 μM MG132 and then centrifuged for 10 min at 12,000 × g. Pure supernatant was used to obtain nuclear proteins.To isolate nuclear proteins from tobacco leaves, nuclei were first isolated from leaves, after which nuclear proteins were isolated using a plant nuclei isolation/extraction kit CelLyticTo extract cytosolic proteins from tobacco leaves, fresh leaves were homogenized with homogenization buffer ). After removal of cell debris by centrifugation for 10 min at 500 × g, sequential centrifugations of the supernatant at higher speeds were performed. Crude nuclear and chloroplast fractions were removed at 1,000 × g for 15 min; mitochondrial fraction was removed at 20,000 × g for 15 min; final supernatant was harvested to obtain the cytosolic proteins.Pisum sativum Toc 75 POTRA domain 3)  was used. We employed primary antibodies for RubisCO protein, ubiquitin, and cellular proteins. Other primary antibodies were as follows: anti-RbcL (Agrisera), anti-His3 (Agrisera), and anti-actin (Agrisera).Proteins  were separated by 4–12% Bis-Tris Plus . Western blotting was performed using a Mini gel tank and Mini Blot Module  according to the manufacturer’s protocol. Proteins were transferred onto iBlot 2 NC Regular Stacks , after which blots were blocked using iBind Cards  according to the manufacturer’s instructions. NPR1-GFP proteins were detected by reacting with mouse monoclonal anti-GFP monoclonal antibody  and horseradish peroxidase-conjugated secondary antibody . Bands were visualized using SuperSingnal West Substrate Working Solution  on X-ray film. To identify TOC proteins in the chloroplast outer envelope membrane, polyclonal rabbit antibody of Toc75–3 (22. Dinitrophenylhydrazine (DNP) was added to samples to derive carbonyl groups from the protein side chains. Derivatized samples were separated by electrophoresis, as described above. Western blot analysis was performed using 2,4-DNP antibody (1:150). DNP signals in integrated intensity of each lane were quantified by densitometry using ImageJ software and normalized to the total protein value of the WT 0 h control, which was set as 1.Oxidized proteins were detected using an OxyBlot protein oxidation detection kit  according to the manufacturer’s protocols2; 5 mM glucose; 1.5 M MES, pH 5.7)60. After gentle centrifugation (5 min at 80 g), protoplasts floating at the interface were collected, washed with W5 (3/1 v/v), pelleted by centrifugation (10 min at 80 g), and resuspended in W5 solution. After stabilization of protoplasts in ice for 30 min, protoplasts at a density of 106/ml were used for further transient transformation.Mesophyll protoplasts from tobacco leaves or Arabidopsis WT leaves were isolated using protoplast extract enzyme solution (pH 5.7) consisting of 1% cellulose R-10 and 0.25% marcerozyme R-10. Leaf slices were transferred to a Petri dish containing enzyme solution and then incubated in the dark for 12 h at 25 °C. After incubation, the enzyme solution was discarded by mesh (10 mm) filtration, after which the cells were overlaid with 1 ml of W5 buffer . These protoplasts were monitored with a confocal microscope.Protoplasts (300 µl) in W5 buffer were pipetted gently into a 0.4 cm pre-chilled electroporation cuvette, and 50 µg of DNA constructs in 10 µl of TE buffer was added. Electroporation was performed using the Gene Pulser Xcell System . Electroporation was carried out with 160 V/960 μF (voltage/capacitance), according to the manufacturer’s instructions. After electroporation, the cuvette was chilled on ice for 10 min, after which protoplasts were transferred to a conical tube using a glass Pasteur pipette with addition of 500 μl of K3 media ) containing MG115, MG132, and plant protease inhibitor cocktail . Protein lysates (30 µg) were precleared with 50 µl of sheep anti-rabbit magnetic beads in a microcentrifuge tube at room temperature for 1 h with gentle rotation. To the precleared lysate, 5% NGS  in PBS was added for blocking, after which primary anti-GFP antibody diluted in PBS was added to a final concentration of 0.2 µg/ml. After incubation of the mixture at 4 °C overnight with gentle rotation, the supernatant was discarded, and the bead mixture was washed in wash buffer  by pipetting gently up and down. Bound proteins were eluted by boiling in 25 µl of 1X SDS sample buffer. The supernatant was analyzed by SDS-PAGE and was used as a substrate for chaperone activity.For the immunoprecipitation of GFP-fused NPR1 proteins, chloroplast stroma proteins and nuclear proteins were separately extracted from 30. MDH was incubated in 50 mM Hepes-KOH (pH 8.0) buffer with various molar ratios of NPR1-GFP recombinant protein purified by immunoprecipitation with GFP antibody from transgenic plants with p35S::NPR1-GFP after salt stress. Aggregation of the substrate was determined under heat denaturation at 45 °C for 30 min by measuring the turbidity at 340 nm using a Shimadzu UV-1601 spectrophotometer .The chaperone activity of NPR1-GFP was assayed by measuring its capacity to suppress thermal aggregation of malate dehydrogenase (MDH) from malic dehydrogenase from porcine heart  as a model substrate61. Statistically significant differences according to t-test between transgenic lines and respective controls at each time point are indicated with one asterisk (*) (P < 0.05) or two asterisks (**) (P < 0.01). Two-way ANOVA was also performed to investigate statistical differences between responses of WT and transgenic lines.All experiments were repeated at least three times with three replicates, and data from one representative experiment are presentedSupplementary information.


Optical see-through automotive head-up displays (HUDs) are a form of augmented reality (AR) that is quickly gaining penetration into the consumer market. Despite increasing adoption, demand, and competition among manufacturers to deliver higher quality HUDs with increased fields of view, little work has been done to understand how best to design and assess AR HUD user interfaces, and how to quantify their effects on driver behavior, performance, and ultimately safety. This paper reports on a novel, low-cost, immersive driving simulator created using a myriad of custom hardware and software technologies specifically to examine basic and applied research questions related to AR HUDs usage when driving. We describe our experiences developing simulator hardware and software and detail a user study that examines driver performance, visual attention, and preferences using two AR navigation interfaces. Results suggest that conformal AR graphics may not be inherently better than other HUD interfaces. We include lessons learned from our simulator development experiences, results of the user study and conclude with limitations and future work. While once the provenance of select academic and government labs, augmented reality (AR) has now been applied in many contexts and delivered over a myriad of hardware technologies. Successes have been documented regarding, for example, smartphone AR on the go , with marketing teams pushing for more advanced AR HUD user interfaces. By 2020, HIS Automotive predicts there will be 9.1 million HUDs sold.must still attend to both the road scene and system information (likely provided via AR HUDs), creating the perfect storm for potentially dangerous and distracting AR HUD interfaces.Moreover, in the very near future, we expect increasingly large AR HUD field of views, affording placement of information in many locations; from windshield-fixed positions to conformal graphics that are perceptually attached to real-world referents. In the same timeframe, we expect an increase in semi-autonomous vehicles where drivers atop the environment, but instead are an integrated part of the environment.While next-generation AR HUDs will provide a fundamentally new driving experience, we currently do not know how to effectively design and evaluate user interfaces (UIs) in this space. With new AR HUDs capable of rendering images over large areas at varying depths, the visual and cognitive separation between graphical and real-world visual stimuli will be increasingly more difficult to quantify. As we move toward widespread use of next-gen AR HUDs in transportation, we need to better understand how to manage UI designs that are not simply How do AR HUD user interfaces that are necessarily visually integrated into a highly dynamic primary task space affect driver performance?” Driving simulators provide a method of rapidly iterating on AR HUD design in realistic driving scenarios without the danger or cost of on-road testing.Without new research capabilities, HUD UI researchers and practitioners are left to base HUD UI design and assessment on current (and dated) understanding of traditional in-vehicle information systems. Common in-vehicle display assessment methods were developed based on data collected in vehicles in the early 2000s  simulator hardware, (2) optical see-through AR displays, and (3) software to realize conformal graphics for driver-vehicle interfaces. For more information about driving simulation in general , see a comprehensive handbook , a wide range of driving simulator hardware has been used in empirical studies on AR applications depending upon the research questions addressed. The lowest fidelity settings are often a combination of desktop computers, monitors and game controllers  are easily detected by the human visual system effectively rendering the potential benefits of conformal graphics disconcerting, distracting or confusing. Until such ability to “lock” conformal AR graphics into the near and far driving scene is established, the use of driving simulators designed specifically to study the effects of AR graphics on driver performance and behavior, such as our system presented herein, is critically needed. It should be noted that while these technical tracking capabilities advance, we can still examine, both in simulation and on-road, AR graphic designs that are more forgiving of tracking and pose estimation errors. For example, Wiesner and Klinker  present In sum, when reviewing the literature, it is clear that several researchers have and continue to engage in meaningful AR HUD research using a myriad of hardware, software, and experimental methodologies. From our review, and to the best of our knowledge, our AR DriveSim is unique in that it provides several synergistic capabilities including: fully immersive vehicle cab and projection system, physiological capture, and measurement , custom electronics for communications and control of vehicle, synchronization of data streams associated with vehicle dynamics, AR interfaces and driver/passengers, autonomous driving, force-feedback to the steering wheel, integrated after-market head-up display, and the ability to render full color, animated, conformal AR graphics.AR DriveSim). From the onset, we established several guiding principles. (1) Embed an actual vehicle cab into a wide field of view 3D projection space  EmpAt its core, the AR DriveSim is a projection-based, monoscopic virtual environment, whereby users “locomote” the environment as a driver of an automobile. In our system, the VR content is provided via MiniSim, a 3D driving simulator software developed at the University of Iowa's National Advanced Driving Simulator research center. MiniSim 2.2 executes on a desktop computer with an Intel Core i7 processing running @ 3.70 GHz, with 64 gigabytes of DDR4 RAM running Windows 10. The driving scene is rendered by a PNY NVIDIA Quadro P4000 graphics card and projected via DisplayPort @ 1,920 × 1,200 using three (warped and blended) Epson Powerlite Pro G6900WU NL projectors. In this hardware configuration, MiniSim provides smooth rendering of up to about 1 million triangles at 60 frames per second. We route these three main forward views through Tripp Lite hardware to mirror the viewports onto three desktop monitors  to proviFor the projection surface, we mounted a professional grade lace-and-grommet screen by Draper that is 93″ high by 360″ long, onto a custom U-shaped, curved frame (73″ inch radius). The projection screen uses a Contrast Gray XH800E smooth gray viewing surface that provides enhanced color contrast and black levels, and is especially useful for our application that uses three projectors with high lumen output. The frame consists of 1½ inch rolled aluminum tubing at both the top and bottom, with 1 × 1 square aluminum tubing structural uprights spaced approximately every 2′.The centerpiece of our driving simulator is the front half of a 2014 Mini Cooper automobile. The vehicle was donated from a major car insurance company that kindly removed the engine and transmission prior to delivery. Once delivered, we tested the electrical components and then completely disassembled the vehicle, including all trim, seats, airbags, dash components, and more until just the frame remained. The back half of the cab was removed and discarded, and the top half of the remaining cab was temporarily removed. The two cab halves were relocated into a lab, where the back-end of the bottom half was mounted on a frame with casters . The top half was the reattached and we then reassembled all the previously removed components (from supporting sub-structures to finished trim pieces) and tested the reassembled vehicle electrical systems.We then incorporated additional displays to support side view mirrors, rear view mirrors, digital instrument panel, and flexible center-stack displays. Specifically, we added three Lilliput 7-inch USB LCD video monitors (800 × 480) connected via powered USB hubs and DisplayLink software to serve as side view mirrors and customizable digital instrument panel . We placLastly, we added a suite of additional equipment to assist in capturing participant behavior. A set of three Axis P1204 3.7 mm mini HD covert pinhole network cameras were placed (1) on the rear-view mirror (facing the participants face), (2) in the driver footwell , and (3) on the center of the cab ceiling pointing at participants' hands on the steering wheel. The cameras are connected to the NOLDUS Observational Suite, which affords synchronized video across the three IP cameras as well as with a direct digital video feed of the driving scene from the drivsim computer. The AR DriveSim also contains both SMI ETG 60 Hz and Tobii Pro Glasses 2 100 Hz wireless eye tracking glasses with forward looking scene camera that allows us to carefully assess drivers' gaze allocation; an especially critical capability for understanding how AR HUD interface designs affect drivers' visual attention. We capture physiological measures of driver workload using a Mio LINK heart rate monitor to capture heart rate variability  bus so that we could leverage existing high-speed control data streams. A To facilitate communication between the Mini Cooper and MiniSim software, we integrated a single board computer (SBC), microcontroller and custom control board to collect and send CAN bus messages, analog voltages from several custom-installed linear potentiometers, and a few OEM sensors .The microcontroller is used to manage the low-complexity, highly-repetitive tasks such as receiving CAN bus messages and reading the analog voltages from the various sensors. We used a Teensy 3.5 because of the built-in CAN bus receiver function, a high number of digital and analog general-purpose input/output, and the flexibility of several protocols for communicating with other systems. The more complex functions of the interface system are managed by a Linux-based SBC that receives parameters from the microcontroller, formats and scales them as needed, and finally composes and sends the data as UDP packets across wired Ethernet to the MiniSim computer. We initially utilized an Arduino Yún as the SBC but following a serial communications issue, we switched to an Intel Galileo Gen2.The Teensy uses the CAN bus interface to access control data such as steering wheel position data, button presses, etc. We installed three linear potentiometers to measure the position of the accelerator pedal, brake pedal, and automatic gearshift position. Each of these parameters is linearly scaled to single byte-sized values and transferred over a serial connection between the Teensy and the Galileo. Upon starting, a python control script stored on the Galileo begins a handshake exchange with the Teensy to establish common timing for the communication scheme. Once communication is started between the two devices, the SBC determines the timing of the transmissions by transmitting a single byte to the Teensy. In response, the Teensy transmits all the steering and position values it has received from the sensors and CAN bus via a two-wire serial connection at 115,200 bps. Once received, the Galileo linearly rescales these values per the MiniSim specifications and packages them into a UDP packet. Testing indicates that this custom interface system reliably transmits 100 packets per second. Although we have not formally measured the end-to-end latency, we expect it to be minimal given (1) MiniSim parses incoming UDP data at 60 Hz, and (2) our own empirical observation.The Teensy communicates with the Cooper's CAN bus using the FlexCAN library  accessible on the MiniSim computer that utilizes a secure shell to issue commands to the Galileo. The control processes are run in the background of the Galileo to provide robustness in the event of a timeout of the secure shell session or other issue. By providing a simple GUI to the communication layer, all researchers regardless of computing background can easily launch and monitor communications between the Mini Cooper, its microcontrollers and simulation software.We also added a Logitech G27 game-based racing wheel and pedals to not only assist in driving scenario development and testing, but more importantly, to allow for Wizard of Oz autonomous driving studies . The aforementioned Python GUI allows researchers to switch between Mini Cooper controls  and game controller .To support our research on the effects of AR interfaces on driver performance and behavior, we integrated a Pioneer Carrozzeria Cyber Navi Head-up display. The Cyber Navi is an optical see-through, fixed focal length (~3 m) laser-based display designed to be mounted on the interior roof in place of a sun visor. We mounted the HUD on a rail along the interior roof of the Mini Cooper so that it can be positioned at varying distances (8–24 inches) from the driver's eyepoint. According to the manufacturer, the Cyber Navi supports a ~17° horizontal field of view, which is consistent with our experiences calibrating the HUD image to the MiniSim driving scene.2 and has an ambient light sensor and automatic dimming capability. The automatic dimming however created color-rendering issues in our simulation environment; at low light levels  the HUD not only dims but also has a strong color bias toward green. That is, white graphics appear green at low lighting levels. To remedy this, we mounted a single LED on a potentiometer directly in front the HUD light sensor. When the LED is lit, the HUD adjusts by creating brighter images resulting in good color rendering. We then applied 20% visible light transmission tinting to the lens to better match the luminance of the HUD graphics to the projected driving scene.As a laser-based display, the Cyber Navi can produce bright images at 12,000 cd/mGenerally speaking, the HUD can render a VGA video source from any VGA-compatible computer and software. This is convenient, as we have successfully conducted user studies using PowerPoint to render 2D screen-fixed text and symbols to assess driver distraction and visual attention with varying HUD positions and UI complexity . In order to present AR graphics, we transmit MiniSim's simulated vehicle position and orientation within the scene. This data is then used to continuously update the position and orientation of the X3D camera.Depending on the nature of the data output by the traffic controller, it may need to be transformed to meet the specification of the scene graph component. To meet X3D's pose specifications, own-vehicle coordinates in MiniSim must be negated along the z-axis. MiniSim's yaw, pitch, and roll values are then used to generate a single rotation vector and magnitude. This transformed data is used to match the pose of X3D's viewpoint to that of the driver within the simulation. This means presentation of conformal AR HUD graphics is defined solely by X3D's viewpoint pose relative to MiniSim's scene.Timing of AR HUD graphics' behavior is done through the use of MiniSim's road pad trigger events which, when driven over by participants, generate event specific network data traffic. For example, in the user study presented below, road pad triggers create data packets that inform the AR HUD software that the driver has encountered an augmented driving segment, and consequently begin rendering the desired AR HUD graphics. The data selected to inform the behavior of conformal graphics is adaptable as a callback mechanism to launch procedures defined in the AR HUD scene graph component. The MiniSim route table can also be configured to send position and orientation data on the nearest 20 dynamic scene objects . Such information can also be used to render real-time conformal graphics such visual pedestrian alerts and labels for nearby traffic.For reference, it should be noted that for the study presented below, we were able to render conformal AR HUD graphics using X3D on a fairly small computer: Intel i5 2,400 s @ 2.5 ghz, 4 gigs ram, Ubuntu 14.04 LTS, running CPU graphics. More complicated AR HUD imagery, either in presentation or behavior, would be well-suited for newer computing and graphics hardware.Because the physical HUD position may need to change to accommodate different driver height and seat positions, it is important that a calibration procedure be performed to ensure accurate perceptual registration of conformal graphics to the driving scene. To accomplish this, participants first sit in the driver's seat and position the seat to a comfortable position. We have participants perform a coarse positioning of the AR HUD combiner  such that top and bottom edges of the combiner align with a prepared calibration image projected onto the curved screen. This ensures that the AR HUD is correctly positioned vertically in the scene so that it, for example, covers the roadway.Next, participants check to ensure that conformal AR graphics perceptually appear in the correct location. For this step, we created a simple highway scenario containing a visible horizon and four vehicles parked at known positions along either side of the highway. The AR HUD software draws boxes around each car as defined by a shared absolute coordinate system. Additionally, the software draws lines correlating to the highway's lane markings to the point of convergence as viewed in simulation . By usinAfter building and refining all driving simulator components, we performed a user study to demonstrate the testbed's research capability. We were especially interested in comparing traditional 2D HUD style graphics to conformal AR graphics since a majority of AR work aims to study the effect of conformal graphics on driver/operator performance.Automotive manufacturers are already implementing 2D screen-fixed AR HUD graphics  in vehicles on the road today. These screen-fixed images are used to display a variety of information, including navigation directions. One area garnering much interest with automotive manufacturers is the potential for georeferenced, world-relative graphics that might be “fixed” in a single location in the world, or dynamic, moving relative to the world, but appearing as part of the world. One of the most common use-cases for these world-relative graphics is navigation, as cues within the world can provide drivers with information to help them navigate throughout complex environments. These two types of graphic use the same technology to convey similar information (where to go) in very different ways. For this reason, our purpose with this study was to compare visual attention, driving behaviors, and experience when using two different types of AR HUD navigational graphics: screen-relative and world-relative, both fixed in location.Conformal arrow was rendered on the HUD and appeared as if it was on the road and blue in color. As participants approached the turn, they “drove over” the arrow as if it was part of the road. Screen-fixed displayed turn directions using a 2D arrow rendered on the HUD, oriented left or right as appropriate, and inspired by current navigation systems. The vertical portion of the Screen-fixed arrow filled as participants approached the turn indicating the distance-to-turn.We compared two different navigation display conditions : a confoAfter study approval from Virginia Tech's Institutional Review Board, we recruited participants using flyers posted on campus, shared with faculty, and email listserv postings. In addition, several participants asked permission to share their experience participating with other friends in the area, and some people chose to participate based on these referrals. Interested individuals contacted the researchers via email to ask any questions regarding the study and to schedule the data collection session, if desired. Upon arrival in the lab, participants consented to participate and entered the driving simulator where they were fitted with eye tracking glasses and adjusted the seat to their comfort. They then performed a familiarization drive to get comfortable with driving simulator setting and vehicle dynamics. We instructed them to drive 30 mph and obey all traffic rules and norms including traffic signals. If they exceeded the speed limit by more than 10%, an audible siren sound was presented indicating that they needed to slow down. The familiarization drive lasted for a minimum of 5 min, until they indicated that they were comfortable with driving the simulator vehicle and the researchers also confirmed that they were able to maintain vehicle control while stopping, starting, turning, and driving straight. After the familiarization drive, we calibrated the HUD vertically and horizontally.Participants experienced the navigation display conditions in a series of drives. Each drive took place in a large city and included eight turns: four right turns and four left turns, all of which were cued by the navigation system and lasted between 6 and 12 min. Differences in duration were largely due to individual differences and randomized traffic patterns. In addition, participants were instructed to attend to oncoming traffic and cross traffic while turning and driving throughout the city. Half of the turns  had cross traffic consisting of a platoon of eight vehicles.Throughout the drive, glance behavior and gaze direction was captured via SensoMotoric Instruments (SMI) eye tracking glasses; which recorded audio, forward facing view, and gaze location for each participant. After data collection, we used SMI BeGaze 3.6.40 analysis software to analyze participants' fixation allocation using manually defined areas of interest. We used the Noldus Observation Suite to record video of the forward-looking road scene independent of participants' gaze direction. This video footage was used to identify participants' risk-taking behaviors. After each drive, participants completed a short series of questionnaires which included workload and usability measures.We collected complete data for 22 participants, all of whom had a US driver's license for longer than 1 year . Thirteen males (mean age 20.3 years) and nine females (mean age 20.4 years) participated. On average, participants drove 7,918 miles per year.We conducted a mixed effects linear model which allowed us to account for individual participant differences as a random effect as seen in de Bruin et al. . We analParticipants self-reported workload using NASA-TLX  for participants' glance location and analyzed the AOIs two ways. The first analysis included two AOIs: on- and off-HUD. The purpose of this distinction is to understand how much drivers limit their gaze to looking only through the HUD as opposed to scanning around the scene. The second AOI coding scheme allowed us to better understand participants' scan patterns to driving-relevant areas . Some reConformal resulted in a significantly higher maximum glance duration toward the HUD graphic than Screen-fixed. Conformal also resulted in longer mean HUD graphic glance durations than Screen-fixed. Further, the number of glances toward the HUD Graphic was significantly higher when participants used the Conformal as compared to the Screen-fixed display type. Conformal was associated with a higher percentage of time looking at the HUD Graphic than Screen-fixed. Screen-fixed resulted in a higher percentage of glances around the HUD graphic than did Conformal. There was no significant difference between the percentage of time that participants looked at off-HUD hazards, on-HUD hazards, or at the HUD in general.In summary, because the conformal display was associated with longer average glances, higher maximum glances, higher glance count, and higher percentage of time focused on the HUD graphic specifically, participants showed a tendency to allocate more visual attention to the conformal HUD graphic than the screen-fixed graphic. Conformal was also associated with less time looking at the area around the HUD graphic and no difference in either on-HUD or off-HUD hazards, showing that the increased visual attention toward the conformal graphic did not necessarily impact participants' hazard scanning behaviors.We analyzed driving data for the total duration of time in which each navigation cue  was visible on the HUD (492 feet prior to each of the eight turns). For each turn, we calculated the relevant lateral, longitudinal, and position control metrics for each trial. We then searched each trial for times when the participant's speed was 0.0 mph and marked these as stops. For the first stop after a graphic appeared, we calculated the distance from the stopping location to the beginning of the intersection. p = 0.6776]. Of those that took a gap, there was no effect of display condition on the gap size that participants chose. Thus, the display type did not significantly impact the drivers' risk-taking behavior, nor did any participant crash into another vehicle during turns (or at any other time during the drive).Using the Noldus video recording, we analyzed participants' risk-taking behavior by capturing how many cars out of a platoon of eight vehicles participants allowed to turn (0–8 vehicles) before deciding to make the turn themselves. If participants turned between two platoon vehicles, we also captured the gap size (in feet) of the distance between those two platoon vehicles. Data from four participants was missing due to human error and therefore we were only able to analyze the risk-taking behavior of 18 participants (out of 22). We were unable to analyze an additional five turns in conformal and two turns in screen-fixed due to simulation scenario, but the mix across turn directions was fairly even . Display condition did not impact the number of cars that participants allowed to turn before making a turn [Our user study included 22 participants who experienced both Conformal and Screen-fixed displays while navigating in our AR DriveSim. In this study, the Screen-fixed display was associated with lower workload  and higher usability  than the Conformal display. The difference in these self-reported measures shows that conformal AR graphics are not necessarily a inherently better user experience, and spatially locating directional graphics into the forward roadway can cause more workload in some instances.There were no differences in driving or risk-taking behaviors despite the fact that participants using the Screen-fixed display allocated less visual attention toward the graphic and therefore, presumably allocated more visual attention toward other elements relevant to the driving task. The lack of differences in driving behaviors can be explained in a study like this because we did not include events that were unexpected or unpredictable in our driving scenarios, which might be more likely to differentiate between HUD graphics. Surprise events (unexpected or unpredictable) require rapid responses and drivers using conformal AR HUDs are especially vulnerable to change blindness or display clutter that might hinder drivers particularly in the face of unexpected events because changes in the display may mask real-world changes. Driving measures are not as sensitive as other physiological measures  or animate on the screen, or even migrate between the road and the screen depending the context. With perfect scene geometry, vehicle tracking, and knowledge of road actors, we can examine UIs attached to other moving vehicles, pedestrians, and bicyclists without attempting to orchestrate those actors in an on-road testbed or trying to track them in real time. We can examine how much tracking error could be tolerated in an on-road AR HUD UI, or how to annotate real-world referents that are outside the AR HUD's field of view. Similarly, we can examine how to design AR UIs that can coexist in heavy traffic, where occlusion is likely to occur and creative context-aware designs need to be developed and tested. By instrumenting and actual vehicle cab with sensing devices  as well as center console touch screens, we can further explore in-vehicle interaction techniques for AR in ways that would be less ideal to conduct in a completely virtual simulated driving environment with virtualized AR HUD graphics . Lastly, by using an actual optical see-through HUD  we can examine physiological and cognitive effects of integrating AR displays with driving scenes such as those associated with context switching and focal distance switching  and further examine how these UIs affect driver behavior and performance. The AR DriveSim's capabilities, however, afford Designing, building, wiring, and programming the AR-DriveSim did not come easy, and as such, we provide a list of lessons learned on the process that may be of value to other researchers and practitioners striving to create similar cyber-physical AR testbeds (be it for driving or other AR application domains).Regarding the physical space for a driving simulator, we recommend larger spaces over smaller ones; at least 5 × 7 m. First, a larger room affords larger cabs, which in turn support a wider range of participant sizes. Larger rooms can also better manage the excessive heat generated by the multitude of computers, displays and projectors needed. This is especially important since warm room temperatures can exacerbate simulator sickness. Larger rooms further afford placement of LCD monitors behind the cab to serve optical side mirrors and a more realistic driver experience. Taller ceilings further allow for more flexibility in purchasing and mounting projectors. If possible, ensure that the physical space contains multiple electrical circuits and a dedicated circuit to power the half cab. If the cab's interior blower fan is operational, it will be extremely useful to have the option to run the fan at its highest speed to help minimize motion sickness, although this requires significant current.When seeking a car to use as a half cab, start by identifying cars with well-documented CAN bus IDs. This will expedite the work needed to connect the cab to the driving simulator software. Also, while it was a good idea to request that the engine and transmission be removed prior to delivery, we recommend that the Engine Control Unit remain intact to provide access to additional CAN bus data. Lastly, if CAN bus IDs are not available, do not invest much time working with simple on-board diagnostic readers, as they yield access to a subset of the total CAN bus traffic. Instead borrow or rent a formal automobile diagnostic tool from a repair shop.Within the physical cab, we recommend routing essential cables underneath and behind trim to not only protect the cables but also to increase the quality of place illusion. That is, you want participants to believe they are in an actual driving car, not a wired-up car in a lab. Route cables for displays, IP cameras, communication, and power before completely reassembling the cab. The cab should also have adjustable seats and a robust HUD positioning and calibration process. Participants that are comfortable and have accurate view of AR content will yield higher quality data.If possible, position the cab such that participants entering the driving simulator space enter from the driver's side. This prevents participants from having to navigate the inevitable set of cables and equipment that are present. Along these lines, we recommend that extra care be taken to manage cables by carefully choosing the right lengths and using cable management techniques. This will help minimize trip hazards for participants and experimenters.Regarding the driving simulator software, it is our strong recommendation that researchers avoid the temptation to develop their own driving simulator software unless the software itself is the desired contribution. A complete driving simulator software solution involves much more than VR graphics including for example, the automated collection of SAE-established driving metrics, integration of real-time complex vehicle dynamics, user-friendly graphical scenario authoring tools, and so forth. While MiniSim is the option we have used, there are other commercial and open-source options available .In terms of the AR HUD software, we found that delegating the transformation tasks  to Python helps simplify the experimental X3D/Javascript source code, and also helps more generally with future portability. Also, while there are likely cases where vehicle-relative coordinate system may be useful, we have found that a common absolute coordinate system greatly simplifies implementation for dynamic AR HUD graphics. This is true especially in cases where researchers do not have deep computing skills, because researchers designing scenarios can specify world-coordinates for AR HUD programmers to use on the X3D/Javascript side. Lastly, when animating conformal AR HUD graphics for turn-based navigation scenarios, we have found that single Bezier curves provide adequate definition for single-turns, and may be linked together to define more complex conditions.While there are a handful of inherent limitations of computer-based driving simulation, we present just a few limitations the driving simulator imposes on our ability to conduct AR HUD research. First, it would be difficult to conduct research related to the effects of real-world lighting and color blending on HUD usage. Even if we could luminance-match, for example, a nighttime scenario, it is not trivial to introduce glare from oncoming traffic and other lighting effects. Similarly, studying the usability of AR HUD graphics on driving backgrounds is limited by the resolution, luminance, dynamic range, and contrast of the projected driving scene. Our AR HUD simulator is also not well-positioned to study issues related to depth perception, since the fixed focal plane HUD coincidentally falls at about the same distance as the projected driving scene. We also do not yet have the ability to articulate the cab and present motion-based cues. In sum, the main limitations restrict our ability to study perceptual AR issues related to outdoor HUD usage. Such studies would need to be conducted while driving on a test track, or fixed indoors looking out.We can easily envision near-term future work that examines the role of AR HUDs in autonomous and semi-autonomous driving. Our integration of a game controller as a secondary means to drive positions us nicely to begin this work. The testbed is also well-suited for integration of 3D spatialized audio to complement the visual HUD UIs. Lastly, we have begun to integrate gesture and voice recognition technology so that we may examine rich AR HUD interaction. Such capabilities will allow us to expand our understanding of driver distraction beyond visual attention.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Virginia Tech Human Research Protection Program. The patients/participants provided their written informed consent to participate in this study.JG directed the creation and integration of all AR DriveSim components, designed the user study, and lead authoring of the manuscript. MS led the user study, conducted data analysis for user study, drafted discussion associated with user study, and edited manuscript. KT developed all software needed for the AR DriveSim tested and authored technical content related to software components. HK performed literature review, helped with logistics of setting up the AR DriveSim and edited manuscript. BJ developed all hardware/microcontroller components needed for the AR DriveSim tested, authored technical content related to software components.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.