ABSTRACT
Successful sexual reproduction of plants requires temperature-sensitive processes, and temperature stress sometimes causes developmental asynchrony between male and female reproductive tissues. In Arabidopsis thaliana, self-pollination occurs when the stamen and pistil lengths are aligned in a single flower so that pollens at the stamen tip are delivered to the stigma at the pistil tip. Although intercellular signalling acts in several reproduction steps, how signalling molecules, including secreted peptides, contribute to the synchronous growth of reproductive tissues remains limited. Here, we show that the mutant of the secreted peptide EPIDERMAL PATTERNING FACTOR LIKE 6 (EPFL6), which shows no phenotypes at a moderate temperature, fails in fruit production at a cool temperature due to insufficient elongation of stamens. EPFL6 is expressed in stamen filaments and promotes filament elongation to achieve the alignment of stamen and pistil lengths at a cool temperature. We also found that, at a moderate temperature, all EPFL6-subfamily genes are required for stamen elongation. Furthermore, we showed that ERECTA (ER), known as a common receptor for EPFL-family peptides, mediates the stamen-pistil growth coordination. Lastly, we provided evidence that modulation of ER activity rescues the reproduction failure caused by insufficient stamen elongation by realigning the stamen and pistil lengths.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Temperature , Pollination , Flowers/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , PeptidesABSTRACT
The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.
Subject(s)
Gene Knockout Techniques/methods , Indoleacetic Acids/chemistry , Plant Proteins/genetics , Proteolysis , Animals , CRISPR-Cas Systems , Cell Line , Chickens , Humans , Oryza/metabolismABSTRACT
In whole-body positron emission tomography (PET)/computed tomography (CT), it is important to optimise the CT radiation dose. We have investigated factors affecting the dose-length product (DLP) of the CT component of whole-body PET/CT and derived equations to predict the DLP. In this retrospective study, 1596 whole-body oncology PET/CT examinations with18F-fluorodeoxyglucose were analysed. Automatic exposure control was used to modulate radiation dose in CT. Considering age, weight, sex, arm position (up, down, one arm up), scan range (up to the mid-thigh or feet), scan mode (spiral or respiratory-triggered nonspiral) and the presence of a metal prosthesis as potential factors, multivariate analysis was performed to identify independent predictors of DLP and to determine equations to predict DLP. DLP values were predicted using the obtained equations, and compared with actual values. Among body size indices, weight best correlated with DLP in examinations performed under the standard imaging conditions (arms: up; scan range: up to the mid-thigh; scan mode: spiral; and no metal prosthesis). Multivariate analysis indicated that weight, arm position, scan range and scan mode were substantial independent predictors; lowering the arms, extending the scan range and using respiratory-triggered imaging, as well as increasing weight, increased DLP. The degree of the DLP increase tended to increase with increasing weight. The DLP values were predicted using equations that considered these parameters were in excellent agreement with the actual values. The DLP for the CT component of whole-body PET/CT is affected by weight, arm position, scan range and scan mode, and can be predicted with excellent accuracy using these factors.
Subject(s)
Positron Emission Tomography Computed Tomography , Tomography, X-Ray Computed , Fluorodeoxyglucose F18 , Positron Emission Tomography Computed Tomography/methods , Radiation Dosage , Retrospective Studies , Tomography, X-Ray Computed/methodsABSTRACT
The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.
Subject(s)
Arabidopsis Proteins/chemistry , F-Box Proteins/chemistry , Gene Expression Regulation, Plant , Indoleacetic Acids/chemistry , Receptors, Cell Surface/chemistry , Arabidopsis/chemistry , Arabidopsis/genetics , Crosses, Genetic , Kinetics , Mutation , Plant Growth Regulators , Plant Roots , Protein Binding , Protein Engineering , Seedlings , Signal Transduction , TransgenesABSTRACT
Auxin regulates diverse aspects of plant growth and development through induction of the interaction between TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX proteins (TIR1/AFBs) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) co-receptor proteins and the subsequent transcriptional regulation. The artificial control of endogenous auxin signaling should enable the precise delineation of auxin-mediated biological events as well as the agricultural application of auxin. To this end, we previously developed a synthetic auxin-receptor pair that consists of 5-(3-methoxyphenyl)-IAA (convexIAA, cvxIAA) and the engineered TIR1 whose phenylalanine at position 79 in the auxin-binding pocket is substituted to glycine (TIR1F79G) (concaveTIR1, ccvTIR1). This synthetic auxin-receptor pair works orthogonally to natural auxin signaling in transgenic plants harboring the engineered TIR1 by exogenous application of 5-(3-methoxyphenyl)-IAA, and has potential to be utilized as novel agricultural/horticultural tools. In the present study, we report an improved version of the synthetic cvxIAA-ccvTIR1 pair such that synthetic IAA can act at lower concentrations. Using a yeast two-hybrid system, we screened various 5-substituted IAAs and identified 5-adamantyl-IAA, named pico_cvxIAA, which mediates interaction of TIR1F79G and IAA3 proteins at a 1,000-fold lower concentration than the original version, 5-(3-methoxyphenyl)-IAA. Furthermore, we found that TIR1F79A interacts with IAA3 protein in the presence of picomolar concentrations of 5-adamantyl-IAA, 10,000-fold lower than our prototype version of the cvxIAA-ccvTIR1 pair. In addition, pull-down assays confirmed that 5-adamantyl-IAA mediates in vitro interaction of TIR1F79A and IAA7-DII peptides at lower concentrations. The improved synthetic IAA-TIR1 pair with high affinity would be beneficial for basic science as well as for practical use in agriculture/horticulture.
Subject(s)
Arabidopsis Proteins/metabolism , F-Box Proteins/metabolism , Indoleacetic Acids/metabolism , Receptors, Cell Surface/metabolism , Nuclear Proteins/metabolism , Protein BindingABSTRACT
Buerger's disease presents a difficult challenge, with its uncertain etiology and lack of a standardized therapy, and is often refractory to treatment. In the present report, we have described the case of a 43-year-old male with Buerger's disease, a refractory ulcer on his right great toe, and severe pain at rest. We obtained favorable results using a hybrid therapy that combined distal bypass with a free temporoparietal fascial flap. Postoperatively, the flap healed, and the patient's pain resolved. After more than 3 years of follow-up, he had had no recurrence of the foot ulceration, and the patency of the bypass graft had been confirmed using Doppler flowmetry. We believe that the hybrid therapy we devised for the present patient (ie, combined distal bypass with a free temporoparietal fascial flap) is an effective treatment of Buerger's disease with pedal ulceration. The therapeutic strategies for this condition should not adhere to a single treatment modality; rather, they should combine available treatment modalities according to each individual patient's condition.
Subject(s)
Blood Vessel Prosthesis Implantation , Foot Ulcer/surgery , Peripheral Arterial Disease/surgery , Surgical Flaps , Adult , Hallux , Humans , Limb Salvage , Male , Microsurgery , Saphenous Vein/transplantation , Surgical Flaps/blood supplyABSTRACT
We investigated the effect of post-labeling delay (PLD) on the evaluation of brain tumor blood flow using arterial spin labeling (ASL) magnetic resonance (MR) imaging to assess the need for imaging with two PLDs. Retrospective analysis was conducted on 63 adult patients with brain tumors who underwent contrast-enhanced MR imaging including ASL imaging with PLDs of both 1525 and 2525 ms on a 1.5 T or 3 T MR unit. Blood flow was estimated in the tumors and normal-appearing brain parenchyma, and tumor blood flow was normalized by parenchymal flow. Estimates of tumor blood flow, parenchymal flow, and normalized tumor flow showed no statistically significant differences between PLDs of 1525 and 2525 ms. Close correlations between different PLDs were found, with the closest correlation for normalized tumor flow. These results were similarly observed for the 1.5 T and 3 T units. The blood flow estimates obtained using ASL MR imaging in patients with brain tumors were highly concordant between PLDs of 1525 and 2525 ms, irrespective of the magnetic field strength. It is indicated that imaging with a single, standard PLD is acceptable for ASL assessment of brain tumor perfusion and that additional imaging with a long PLD is not required.
Subject(s)
Brain Neoplasms , Magnetic Resonance Imaging , Adult , Humans , Spin Labels , Retrospective Studies , Magnetic Resonance Imaging/methods , Brain Neoplasms/pathology , Magnetic Resonance SpectroscopyABSTRACT
Plants retain the ability to generate a pluripotent tissue called callus by dedifferentiating somatic cells. A pluripotent callus can also be artificially induced by culturing explants with hormone mixtures of auxin and cytokinin, and an entire body can then be regenerated from the callus. Here we identified a pluripotency-inducing small compound, PLU, that induces the formation of callus with tissue regeneration potency without the external application of either auxin or cytokinin. The PLU-induced callus expressed several marker genes related to pluripotency acquisition via lateral root initiation processes. PLU-induced callus formation required activation of the auxin signaling pathway though the amount of active auxin was reduced by PLU treatment. RNA-seq analysis and subsequent experiments revealed that Heat Shock Protein 90 (HSP90) mediates a significant part of the PLU-initiated early events. We also showed that HSP90-dependent induction of TRANSPORT INHIBITOR RESPONSE 1, an auxin receptor gene, is required for the callus formation by PLU. Collectively, this study provides a new tool for manipulating and investigating the induction of plant pluripotency from a different angle from the conventional method with the external application of hormone mixtures.
ABSTRACT
Differentiation of specialized cell types requires precise cell-cycle control. Plant stomata are generated through asymmetric divisions of a stem-cell-like precursor followed by a single symmetric division that creates paired guard cells surrounding a pore. The stomatal-lineage-specific transcription factor MUTE terminates the asymmetric divisions and commits to differentiation. However, the role of cell-cycle machineries in this transition remains unknown. We discover that the symmetric division is slower than the asymmetric division in Arabidopsis. We identify a plant-specific cyclin-dependent kinase inhibitor, SIAMESE-RELATED4 (SMR4), as a MUTE-induced molecular brake that decelerates the cell cycle. SMR4 physically and functionally associates with CYCD3;1 and extends the G1 phase of asymmetric divisions. By contrast, SMR4 fails to interact with CYCD5;1, a MUTE-induced G1 cyclin, and permits the symmetric division. Our work unravels a molecular framework of the proliferation-to-differentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for stomatal-lineage identity.