Publications by authors named "Masao Tasaka"

103 Publications

Establishment of the Embryonic Shoot Meristem Involves Activation of Two Classes of Genes with Opposing Functions for Meristem Activities.

Int J Mol Sci 2020 Aug 15;21(16). Epub 2020 Aug 15.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Nara 630-0192, Japan.

The shoot meristem, a stem-cell-containing tissue initiated during plant embryogenesis, is responsible for continuous shoot organ production in postembryonic development. Although key regulatory factors including genes are responsible for stem cell maintenance in the shoot meristem, how the onset of such factors is regulated during embryogenesis is elusive. Here, we present evidence that the two genes and together with the two other regulatory genes and are functionally important downstream genes of and , which are a redundant pair of genes that specify the embryonic shoot organ boundary. Combined expression of with any of , , and can efficiently rescue the defects of shoot meristem formation and/or separation of cotyledons in double mutants. In addition, and are also required for the activation of , a cytochrome P450-encoding gene known to restrict organ production, and counteracts in the promotion of meristem activity, providing a possible balancing mechanism for shoot meristem maintenance. Together, these results establish the roles for and in coordinating the activation of two classes of genes with opposite effects on shoot meristem activity.
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http://dx.doi.org/10.3390/ijms21165864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461597PMC
August 2020

Lateral root initiation requires the sequential induction of transcription factors LBD16 and PUCHI in Arabidopsis thaliana.

New Phytol 2019 10 10;224(2):749-760. Epub 2019 Aug 10.

Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Kobe, 657-8501, Japan.

Lateral root (LR) formation in Arabidopsis thaliana is initiated by asymmetric division of founder cells, followed by coordinated cell proliferation and differentiation for patterning new primordia. The sequential developmental processes of LR formation are triggered by a localized auxin response. LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), an auxin-inducible transcription factor, is one of the key regulators linking auxin response in LR founder cells to LR initiation. We identified key genes for LR formation that are activated by LBD16 in an auxin-dependent manner. LBD16 targets identified include the transcription factor gene PUCHI, which is required for LR primordium patterning. We demonstrate that LBD16 activity is required for the auxin-inducible expression of PUCHI. We show that PUCHI expression is initiated after the first round of asymmetric cell division of LR founder cells and that premature induction of PUCHI during the preinitiation phase disrupts LR primordium formation. Our results indicate that LR initiation requires the sequential induction of transcription factors LBD16 and PUCHI.
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http://dx.doi.org/10.1111/nph.16065DOI Listing
October 2019

Mitochondrial Pyruvate Dehydrogenase Contributes to Auxin-Regulated Organ Development.

Plant Physiol 2019 06 20;180(2):896-909. Epub 2019 Mar 20.

College of Life Sciences, Fujian Agriculture and Forestry University, No.15 Shangxiadian Road, Cangshan District, Fuzhou City, Fujian 350002, China

Pyruvate dehydrogenase is the first enzyme (E1) of the PDH complex (PDC). This multienzyme complex contains E1, E2, and E3 components and controls the entry of carbon into the mitochondrial tricarboxylic acid cycle to enable cellular energy production. The E1 component of the PDC is composed of an E1α catalytic subunit and an E1β regulatory subunit. In Arabidopsis (), there are two mitochondrial E1α homologs encoded by () and (), and one mitochondrial E1β homolog. Although IAR4 was reported to be involved in auxin conjugate sensitivity and auxin homeostasis in root development, its precise role remains unknown. Here, we provide experimental evidence that mitochondrial PDC E1 contributes to polar auxin transport during organ development. We performed genetic screens for factors involved in cotyledon development and identified an uncharacterized mutant, (). encodes a mitochondrial PDC E1β subunit that can form both a homodimer and a heterodimer with IAR4. The mutation impaired MAB1 homodimerization, reduced the abundance of IAR4 and IAR4L, weakened PDC enzymatic activity, and diminished mitochondrial respiration. A metabolomics analysis showed significant changes in metabolites including amino acids in and, in particular, identified an accumulation of Ala. These results suggest that MAB1 is a component of the Arabidopsis mitochondrial PDC E1. Furthermore, in mutants and seedlings where the TCA cycle was pharmacologically blocked, we found reduced abundance of the PIN-FORMED (PIN) auxin efflux carriers, possibly due to impaired PIN recycling and enhanced PIN degradation in vacuoles. Therefore, we suggest that induces defective polar auxin transport via metabolic abnormalities.
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http://dx.doi.org/10.1104/pp.18.01460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548247PMC
June 2019

Polar vacuolar distribution is essential for accurate asymmetric division of zygotes.

Proc Natl Acad Sci U S A 2019 02 16;116(6):2338-2343. Epub 2019 Jan 16.

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Aichi, Japan;

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that (), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In , large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.
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http://dx.doi.org/10.1073/pnas.1814160116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369786PMC
February 2019

Lateral Inhibition by a Peptide Hormone-Receptor Cascade during Arabidopsis Lateral Root Founder Cell Formation.

Dev Cell 2019 01 20;48(1):64-75.e5. Epub 2018 Dec 20.

Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Kobe 657-8501, Japan. Electronic address:

In plants, the position of lateral roots (LRs) depends on initiation sites induced by auxin. The domain of high auxin response responsible for LR initiation stretches over several cells, but only a pair of pericycle cells (LR founder cells) will develop into LRs. In this work, we identified a signaling cascade controlling LR formation through lateral inhibition. It comprises a peptide hormone TARGET OF LBD SIXTEEN 2 (TOLS2), its receptor RLK7, and a downstream transcription factor PUCHI. TOLS2 is expressed at the LR founder cells and inhibits LR initiation. Time-lapse imaging of auxin-responsive DR5:LUCIFERASE reporter expression revealed that occasionally two pairs of LR founder cells are specified in close proximity even in wild-type and that one of them exists only transiently and disappears in an RLK7-dependent manner. We propose that the selection of LR founder cells by the peptide hormone-receptor cascade ensures proper LR spacing.
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http://dx.doi.org/10.1016/j.devcel.2018.11.031DOI Listing
January 2019

ERECTA-family genes coordinate stem cell functions between the epidermal and internal layers of the shoot apical meristem.

Development 2018 01 8;145(1). Epub 2018 Jan 8.

Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan

The epidermal cell layer and the tissues that lie underneath have different intrinsic functions during plant development. The stem cells within the shoot apical meristem (SAM) that give rise to aerial structures are located in the epidermal and internal tissue layers. However, our understanding of how the functions of these stem cells are coordinated across tissue layers so stem cells can behave as a single population remains limited. WUSCHEL (WUS) functions as a master regulator of stem cell activity. Here, we show that loss of function in the ERECTA (ER)family receptor kinase genes can rescue the mutant phenotype of plants (loss of stem cells), as demonstrated by the reinstated expression of a stem cell marker gene in the SAM epidermis. Localized expression in the epidermis can suppress the SAM phenotype caused by loss of ER-family activity. Furthermore, the CLAVATA3- and cytokinin-induced outputs, which contribute to stem cell homeostasis, are dysfunctional in a tissue layer-specific manner in ER-family mutants. Collectively, our findings suggest that the ER family plays a role in the coordination of stem cell behavior between different SAM tissue layers.
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http://dx.doi.org/10.1242/dev.156380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825868PMC
January 2018

The Arabidopsis LAZY1 Family Plays a Key Role in Gravity Signaling within Statocytes and in Branch Angle Control of Roots and Shoots.

Plant Cell 2017 Aug 1;29(8):1984-1999. Epub 2017 Aug 1.

Graduate School of Bioagricultural Sciences, Nagoya University, Furocho, Chikusa, Nagoya 464-8601, Japan

During gravitropism, the directional signal of gravity is perceived by gravity-sensing cells called statocytes, leading to asymmetric distribution of auxin in the responding organs. To identify the genes involved in gravity signaling in statocytes, we performed transcriptome analyses of statocyte-deficient mutants and found two candidates from the LAZY1 family, / () and // We showed that , , and a paralog are redundantly involved in gravitropism of the inflorescence stem, hypocotyl, and root. Mutations of genes affected early processes in gravity signal transduction without affecting amyloplast sedimentation. Statocyte-specific expression of genes rescued the mutant phenotype, suggesting that genes mediate gravity signaling in statocytes downstream of amyloplast displacement, leading to the generation of asymmetric auxin distribution in gravity-responding organs. We also found that mutations reversed the growth angle of lateral branches and roots. Moreover, expression of the conserved C-terminal region of LZY proteins also reversed the growth direction of primary roots in the mutant background. In lateral root tips of multiple mutants, asymmetric distribution of PIN3 and auxin response were reversed, suggesting that genes regulate the direction of polar auxin transport in response to gravity through the control of asymmetric PIN3 expression in the root cap columella.
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http://dx.doi.org/10.1105/tpc.16.00575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590491PMC
August 2017

ERECTA-family receptor kinase genes redundantly prevent premature progression of secondary growth in the Arabidopsis hypocotyl.

New Phytol 2017 Mar 28;213(4):1697-1709. Epub 2016 Nov 28.

Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.

Secondary growth is driven by continuous cell proliferation and differentiation of the cambium that acts as vascular stem cells, producing xylem and phloem to expand vascular tissues laterally. During secondary growth of hypocotyls in Arabidopsis thaliana, the xylem undergoes a drastic phase transition from a parenchyma-producing phase to a fiber-producing phase at the appropriate time. However, it remains to be fully elucidated how progression of secondary growth is properly controlled. We focused on phenotypes of hypocotyl vasculatures caused by double mutation in ERECTA (ER) and ER-LIKE1 (ERL1) receptor-kinase genes to elucidate their roles in secondary growth. ER and ERL1 redundantly suppressed excessive radial growth of the hypocotyl vasculature during secondary growth. ER and ERL1 also prevented premature initiation of the fiber differentiation process mediated by the NAC SECONDARY WALL THICKENING PROMOTING FACTORs in the hypocotyl xylem. Upon floral transition, the hypocotyl xylem gained a competency to respond to GA in a BREVIPEDICELLUS-dependent manner, which was a prerequisite for fiber differentiation. However, even after the floral transition, ER and ERL1 prevented precocious initiation of the GA-mediated fiber formation. Collectively, our findings reveal that ER and ERL1 redundantly prevent premature progression of sequential events in secondary growth.
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http://dx.doi.org/10.1111/nph.14335DOI Listing
March 2017

Impact of erecta mutation on leaf serration differs between Arabidopsis accessions.

Plant Signal Behav 2016 12;11(12):e1261231

a Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University , Furo-cho, Chikusa-ku , Nagoya , Japan.

Serrations or teeth of plant leaves are a morphological trait regulated genetically and environmentally. Very recently, it has been reported that the receptor kinases encoded by three ERECTA (ER)-family genes, ER, ER-LIKE1 (ERL1) and ERL2, redundantly play a role in tooth growth in Arabidopsis thaliana. In the report, Columbia (Col) accession was used for analyses, where none of the signal mutant of the ER-family genes exhibited serration defects. The toothless, smooth leaf margin phenotype was evident only when two out of the three ER-family genes were lost. Interestingly, it has been widely recognized that the Arabidopsis accession Landsberg erecta (L.er), which carries a loss-of-function mutation in ER, develops round leaves with smaller leaf teeth. Here, we show that the functional ER transgene promotes the tooth growth in L.er to the level of Col, indicating that the er mutation in L.er is likely responsible for the reduced growth of leaf teeth. This suggests that er single mutation affects tooth growth in a different manner between Col and L.er backgrounds, though the molecular basis for this background-dependent effect remains to be addressed.
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http://dx.doi.org/10.1080/15592324.2016.1261231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225933PMC
December 2016

Isolation of New Gravitropic Mutants under Hypergravity Conditions.

Front Plant Sci 2016 29;7:1443. Epub 2016 Sep 29.

Graduate School of Bioagricultural Sciences, Nagoya UniversityNagoya, Japan; CREST, Japan Science and Technology AgencyTokyo, Japan.

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the () mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the background. Using the new screening system, we successfully isolated six () mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in .
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http://dx.doi.org/10.3389/fpls.2016.01443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040707PMC
September 2016

A Secreted Peptide and Its Receptors Shape the Auxin Response Pattern and Leaf Margin Morphogenesis.

Curr Biol 2016 09 1;26(18):2478-2485. Epub 2016 Sep 1.

Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. Electronic address:

Secreted peptides mediate intercellular communication [1, 2]. Several secreted peptides in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family regulate morphogenesis of tissues, such as stomata and inflorescences in plants [3-15]. The biological functions of other EPFL family members remain unknown. Here, we show that the EPFL2 gene is required for growth of leaf teeth. EPFL2 peptide physically interacts with ERECTA (ER) family receptor-kinases and, accordingly, the attenuation of ER family activities leads to formation of toothless leaves. During the tooth growth process, responses to the phytohormone auxin are maintained at tips of the teeth to promote their growth [16-19]. In the growing tooth tip of epfl2 and multiple er family mutants, the auxin response becomes broader. Conversely, overexpression of EPFL2 diminishes the auxin response, indicating that the EPFL2 signal restricts the auxin response to the tooth tip. Interestingly, the tip-specific auxin response in turn organizes characteristic expression patterns of ER family and EPFL2 by enhancing ER family expression at the tip while eliminating the EPFL2 expression from the tip. Our findings identify the novel ligand-receptor pairs promoting the tooth growth, and further reveal a feedback circuit between the peptide-receptor system and auxin response as a mechanism for maintaining proper auxin maxima during leaf margin morphogenesis.
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http://dx.doi.org/10.1016/j.cub.2016.07.014DOI Listing
September 2016

Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

Proc Natl Acad Sci U S A 2016 Jun 23;113(23):6562-7. Epub 2016 May 23.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan;

Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.
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http://dx.doi.org/10.1073/pnas.1600739113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988599PMC
June 2016

Efficient In Planta Detection and Dissection of De Novo Mutation Events in the Arabidopsis thaliana Disease Resistance Gene UNI.

Plant Cell Physiol 2016 Jun 25;57(6):1123-32. Epub 2016 Mar 25.

Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601 Japan

Plants possess disease resistance (R) proteins encoded by R genes, and each R protein recognizes a specific pathogen factor(s) for immunity. Interestingly, a remarkably high degree of polymorphisms in R genes, which are traces of past mutation events during evolution, suggest the rapid diversification of R genes. However, little is known about molecular aspects that facilitate the rapid change of R genes because of the lack of tools that enable us to monitor de novo R gene mutations efficiently in an experimentally feasible time scale, especially in living plants. Here we introduce a model assay system that enables efficient in planta detection of de novo mutation events in the Arabidopsis thaliana R gene UNI in one generation. The uni-1D mutant harbors a gain-of-function allele of the UNI gene. uni-1D heterozygous individuals originally exhibit dwarfism with abnormally short stems. However, interestingly, morphologically normal stems sometimes emerge spontaneously from the uni-1D plants, and the morphologically reverted tissues carry additional de novo mutations in the UNI gene. Strikingly, under an extreme condition, almost half of the examined population shows the reversion phenomenon. By taking advantage of this phenomenon, we demonstrate that the reversion frequency is remarkably sensitive to a variety of fluctuations in DNA stability, underlying a mutable tendency of the UNI gene. We also reveal that activities of the salicylic acid pathway and DNA damage sensor pathway are involved in the reversion phenomenon. Thus, we provide an experimentally feasible model tool to explore factors and conditions that significantly affect the R gene mutation phenomenon.
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http://dx.doi.org/10.1093/pcp/pcw060DOI Listing
June 2016

Transcriptional regulation of PIN genes by FOUR LIPS and MYB88 during Arabidopsis root gravitropism.

Nat Commun 2015 Nov 18;6:8822. Epub 2015 Nov 18.

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan (Fragrant Hill), Haidian, Beijing 100093, China.

PIN proteins are auxin export carriers that direct intercellular auxin flow and in turn regulate many aspects of plant growth and development including responses to environmental changes. The Arabidopsis R2R3-MYB transcription factor FOUR LIPS (FLP) and its paralogue MYB88 regulate terminal divisions during stomatal development, as well as female reproductive development and stress responses. Here we show that FLP and MYB88 act redundantly but differentially in regulating the transcription of PIN3 and PIN7 in gravity-sensing cells of primary and lateral roots. On the one hand, FLP is involved in responses to gravity stimulation in primary roots, whereas on the other, FLP and MYB88 function complementarily in establishing the gravitropic set-point angles of lateral roots. Our results support a model in which FLP and MYB88 expression specifically determines the temporal-spatial patterns of PIN3 and PIN7 transcription that are closely associated with their preferential functions during root responses to gravity.
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http://dx.doi.org/10.1038/ncomms9822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673497PMC
November 2015

An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

Plant Signal Behav 2016 ;11(3):e1010947

a Graduate School of Science, Kyoto University , Kyoto 606-8502 , Japan.

A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.
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http://dx.doi.org/10.1080/15592324.2015.1010947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4883830PMC
December 2016

Live cell imaging of cytoskeletal and organelle dynamics in gravity-sensing cells in plant gravitropism.

Methods Mol Biol 2015 ;1309:57-69

Institute of Biochemistry and Biology, Plant Physiology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Building 20, 14476, Potsdam-Golm, Germany.

Plants sense gravity and change their morphology/growth direction accordingly (gravitropism). The early process of gravitropism, gravity sensing, is supposed to be triggered by sedimentation of starch-filled plastids (amyloplasts) in statocytes such as root columella cells and shoot endodermal cells. For several decades, many scientists have focused on characterizing the role of the amyloplasts and observed their intracellular sedimentation in various plants. Recently, it has been discovered that the complex sedimentary movements of the amyloplasts are created not only by gravity but also by cytoskeletal/organelle dynamics, such as those of actin filaments and the vacuolar membrane. Thus, to understand how plants sense gravity, we need to analyze both amyloplast movements and their regulatory systems in statocytes. We have developed a vertical-stage confocal microscope that allows multicolor fluorescence imaging of amyloplasts, actin filaments and vacuolar membranes in vertically oriented plant tissues. We also developed a centrifuge microscope that allows bright-field imaging of amyloplasts during centrifugation. These microscope systems provide new insights into gravity-sensing mechanisms in Arabidopsis.
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http://dx.doi.org/10.1007/978-1-4939-2697-8_6DOI Listing
February 2016

Identification of gravitropic response indicator genes in Arabidopsis inflorescence stems.

Plant Signal Behav 2014 ;9(9):e29570

a Graduate School of Bioagricultural Sciences; Nagoya University; Furo-cho, Chikusa-ku, Nagoya, Japan.

Differential organ growth during gravitropic response is caused by differential accumulation of auxin, that is, relative higher auxin concentration in lower flanks than in upper flanks of responding organs. Auxin responsive reporter systems such as DR5::GUS and DR5::GFP have usually been used as indicators of gravitropic response in roots and hypocotyls of Arabidopsis. However, in the inflorescence stems, the reporter systems don't work well to monitor gravitropic response. Here, we aim to certify appropriate gravitropic response indicators (GRIs) in inflorescence stems. We performed microarray analysis comparing gene expression profiles between upper and lower flanks of Arabidopsis inflorescence stems after gravistimulation. Thirty genes showed > 2-fold differentially increased expression in lower flanks at 30 min, of which 19 were auxin response genes. We focused on IAA5 and IAA2 and verified whether they are appropriate GRIs by real-time qRT-PCR analyses. Transcript levels of IAA5 and IAA2 were remarkably higher in lower flanks than in upper flanks after gravistimulation. The biased IAA5 or IAA2 expression is disappeared in sgr2-1 mutant which is defective in gravity perception, indicating that gravity perception process is essential for formation of the biased gene expression during gravitropism. IAA5 expression was remarkably increased in lower flanks at 30 min after gravistimulation, whereas IAA2 expression was gradually decreased in upper flanks in a time-dependent manner. Therefore, we conclude that IAA5 is a sensitive GRI to monitor asymmetric auxin signaling caused by gravistimulation in Arabidopsis inflorescence stems.
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http://dx.doi.org/10.4161/psb.29570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203507PMC
November 2015

Regulation of organ straightening and plant posture by an actin-myosin XI cytoskeleton.

Nat Plants 2015 Mar 23;1(4):15031. Epub 2015 Mar 23.

Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

Plants are able to bend nearly every organ in response to environmental stimuli such as gravity and light(1,2). After this first phase, the responses to stimuli are restrained by an independent mechanism, or even reversed, so that the organ will stop bending and attain its desired posture. This phenomenon of organ straightening has been called autotropism(3) and autostraightening(4) and modelled as proprioception(5). However, the machinery that drives organ straightening and where it occurs are mostly unknown. Here, we show that the straightening of inflorescence stems is regulated by an actin-myosin XI cytoskeleton in specialized immature fibre cells that are parallel to the stem and encircle it in a thin band. Arabidopsis mutants defective in myosin XI (specifically XIf and XIk) or ACTIN8 exhibit hyperbending of stems in response to gravity, an effect independent of the physical properties of the shoots. The actin-myosin XI cytoskeleton enables organs to attain their new position more rapidly than would an oscillating series of diminishing overshoots in environmental stimuli. We propose that the long actin filaments in elongating fibre cells act as a bending tensile sensor to perceive the organ's posture and trigger the straightening system.
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http://dx.doi.org/10.1038/nplants.2015.31DOI Listing
March 2015

The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development.

Front Plant Sci 2014 30;5:165. Epub 2014 Apr 30.

Department of Plant Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology Ikoma, Japan.

Carpel margin meristems (CMMs), a pair of meristematic tissues present along the margins of two fused carpel primordia of Arabidopsis thaliana, are essential for the formation of ovules and the septum, two major internal structures of the gynoecium. Although a number of regulatory factors involved in shoot meristem activity are known to be required for the formation of these gynoecial structures, their direct roles in CMM development have yet to be addressed. Here we show that the CUP-SHAPED COTYLEDON genes CUC1 and CUC2, which are essential for shoot meristem initiation, are also required for formation and stable positioning of the CMMs. Early in CMM formation, CUC1 and CUC2 are also required for expression of the SHOOT MERISTEMLESS gene, a central regulator for stem cell maintenance in the shoot meristem. Moreover, plants carrying miR164-resistant forms of CUC1 and CUC2 resulted in extra CMM activity with altered positioning. Our results thus demonstrate that the two regulatory proteins controlling shoot meristem activity also play critical roles in elaboration of the female reproductive organ through the control of meristematic activity.
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http://dx.doi.org/10.3389/fpls.2014.00165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012194PMC
May 2014

A unique HEAT repeat-containing protein SHOOT GRAVITROPISM6 is involved in vacuolar membrane dynamics in gravity-sensing cells of Arabidopsis inflorescence stem.

Plant Cell Physiol 2014 Apr 30;55(4):811-22. Epub 2014 Jan 30.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan.

Plant vacuoles play critical roles in development, growth and stress responses. In mature cells, vacuolar membranes (VMs) display several types of structures, which are formed by invagination and folding of VMs into the lumenal side and can gradually move and change shape. Although such VM structures are observed in a broad range of tissue types and plant species, the molecular mechanism underlying their formation and maintenance remains unclear. Here, we report that a novel HEAT-repeat protein, SHOOT GRAVITROPISM6 (SGR6), of Arabidopsis is involved in the control of morphological changes and dynamics of VM structures in endodermal cells, which are the gravity-sensing cells in shoots. SGR6 is a membrane-associated protein that is mainly localized to the VM in stem endodermal cells. The sgr6 mutant stem exhibits a reduced gravitropic response. Higher plants utilize amyloplast sedimentation as a means to sense gravity direction. Amyloplasts are surrounded by VMs in Arabidopsis endodermal cells, and the flexible and dynamic structure of VMs is important for amyloplast sedimentation. We demonstrated that such dynamic features of VMs are gradually lost in sgr6 endodermal cells during a 30 min observation period. Histological analysis revealed that amyloplast sedimentation was impaired in sgr6. Detailed live-cell imaging analyses revealed that the VM structures in sgr6 had severe defects in morphological changes and dynamics. Our results suggest that SGR6 is a novel protein involved in the formation and/or maintenance of invaginated VM structures in gravity-sensing cells.
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http://dx.doi.org/10.1093/pcp/pcu020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982123PMC
April 2014

Auxin transport and activity regulate stomatal patterning and development.

Nat Commun 2014 ;5:3090

Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.

Stomata are two-celled valves that control epidermal pores whose spacing optimizes shoot-atmosphere gas exchange. They develop from protodermal cells after unequal divisions followed by an equal division and differentiation. The concentration of the hormone auxin, a master plant developmental regulator, is tightly controlled in time and space, but its role, if any, in stomatal formation is obscure. Here dynamic changes of auxin activity during stomatal development are monitored using auxin input (DII-VENUS) and output (DR5:VENUS) markers by time-lapse imaging. A decrease in auxin levels in the smaller daughter cell after unequal division presages the acquisition of a guard mother cell fate whose equal division produces the two guard cells. Thus, stomatal patterning requires auxin pathway control of stem cell compartment size, as well as auxin depletion that triggers a developmental switch from unequal to equal division.
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http://dx.doi.org/10.1038/ncomms4090DOI Listing
October 2015

MAB4-induced auxin sink generates local auxin gradients in Arabidopsis organ formation.

Proc Natl Acad Sci U S A 2014 Jan 6;111(3):1198-203. Epub 2014 Jan 6.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0192, Japan.

In Arabidopsis, leaves and flowers form cyclically in the shoot meristem periphery and are triggered by local accumulations of the plant hormone auxin. Auxin maxima are established by the auxin efflux carrier PIN-formed1 (PIN1). During organ formation, two distinct types of PIN1 polarization occur. First, convergence of PIN1 polarity in the surface of the meristem creates local auxin peaks. Second, basipetal PIN1 polarization causes auxin to move away from the surface in the middle of an incipient organ primordium, thought to contribute to vascular formation. Several mathematical models have been developed in attempts to explain the PIN1 localization pattern. However, the molecular mechanisms that control these dynamic changes are unknown. Here, we show that loss-of-function in the MACCHI-BOU 4 (MAB4) family genes, which encode nonphototropic hypocotyl 3-like proteins and regulate PIN endocytosis, cause deletion of basipetal PIN1 polarization, resulting in extensive auxin accumulation all over the meristem surface from lack of a sink for auxin. These results indicate that the MAB4 family genes establish inward auxin transport from the L1 surface of incipient organ primordia by basipetal PIN1 polarization, and that this behavior is essential for the progression of organ development. Furthermore, the expression of the MAB4 family genes depends on auxin response. Our results define two distinct molecular mechanisms for PIN1 polarization during organ development and indicate that an auxin response triggers the switching between these two mechanisms.
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http://dx.doi.org/10.1073/pnas.1316109111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903239PMC
January 2014

Identification of EMS-induced causal mutations in Arabidopsis thaliana by next-generation sequencing.

Methods Mol Biol 2014 ;1062:259-70

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan.

Emerging next-generation sequencing (NGS) technologies are powerful tools for the identification of causal mutations underlying phenotypes of interest in Arabidopsis thaliana. Based on a methodology termed bulked segregant analysis (BSA), whole-genome sequencing data are derived from pooled F2 segregants after crossing a mutant to a different polymorphic accession and are analyzed for single nucleotide polymorphisms (SNPs). Then, a genome region spanning the causal mutation site is narrowed down by linkage analysis of SNPs in the accessions used to produce the F1 generation. Next, candidate SNPs for the causative mutation are extracted by filtering the linked SNPs using multiple appropriate criteria. Effects of each candidate SNP on the function of the corresponding gene are evaluated to identify the causal mutation, and its validity is then confirmed by independent criteria. This chapter describes the identification by NGS analysis of causal recessive mutations derived from EMS mutagenesis.
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http://dx.doi.org/10.1007/978-1-62703-580-4_14DOI Listing
April 2014

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope.

Plant J 2013 Nov 14;76(4):648-60. Epub 2013 Oct 14.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan; Department of Botany, University of Wisconsin, Madison, WI, 53706, USA.

The starch-statolith hypothesis proposes that starch-filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so-called 'static' or 'settled' statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom-designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild-type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity-driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity-induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild-type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the 'bottom' of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.
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http://dx.doi.org/10.1111/tpj.12324DOI Listing
November 2013

Regulation of plant vascular stem cells by endodermis-derived EPFL-family peptide hormones and phloem-expressed ERECTA-family receptor kinases.

J Exp Bot 2013 Dec 23;64(17):5335-43. Epub 2013 Jul 23.

WPI-Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.

Plant vasculatures are complex tissues consisting of (pro)cambium, phloem, and xylem. The (pro)cambium serves as vascular stem cells that produce all vascular cells. The Arabidopsis ERECTA (ER) receptor kinase is known to regulate the architecture of inflorescence stems. It was recently reported that the er mutation enhances a vascular phenotype induced by a mutation of TDR/PXY, which plays a significant role in procambial proliferation, suggesting that ER participates in vascular development. However, detailed molecular mechanisms of the ER-dependent vascular regulation are largely unknown. Here, this work found that ER and its paralogue, ER-LIKE1, were redundantly involved in procambial development of inflorescence stems. Interestingly, their activity in the phloem was sufficient for vascular regulation. Furthermore, two endodermis-derived peptide hormones, EPFL4 and EPFL6, were redundantly involved in such regulation. It has been previously reported that EPFL4 and EPFL6 act as ligands of phloem-expressed ER for stem elongation. Therefore, these findings indicate that cell-cell communication between the endodermis and the phloem plays an important role in procambial development as well as stem elongation. Interestingly, similar EPFL-ER modules control two distinct developmental events by slightly changing their components: the EPFL4/6-ER module for stem elongation and the EPFL4/6-ER/ERL1 module for vascular development.
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http://dx.doi.org/10.1093/jxb/ert196DOI Listing
December 2013

Cell biology - building blocks for dynamic development and behaviors.

Curr Opin Plant Biol 2012 Dec 19;15(6):575-7. Epub 2012 Nov 19.

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http://dx.doi.org/10.1016/j.pbi.2012.11.001DOI Listing
December 2012

Mechanism of higher plant gravity sensing.

Am J Bot 2013 Jan 31;100(1):91-100. Epub 2012 Oct 31.

Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama 8916-5, Ikoma, Nara 630-0192, Japan.

Higher plants have developed statocytes, specialized tissues or cells for gravity sensing, and subsequent signal formation. Root and shoot statocytes commonly harbor a number of amyloplasts, and amyloplast sedimentation in the direction of gravity is a critical process in gravity sensing. However, the molecular mechanism underlying amyloplast-dependent gravity sensing is largely unknown. In this review, we mainly describe the molecular basis for the gravity sensing mechanism, i.e., the molecules and their functions involved in amyloplast sedimentation. Several analyses of statocyte images in living plant organs have implied differences in the regulation of amyloplast movements between root and shoot statocytes. Amyloplasts in shoot statocytes display not only sedimentable but upward, saltatory movements, but the latter are rarely observed in root statocytes. A series of genetic studies on shoot gravitropism mutants of Arabidopsis thaliana has revealed that two intracellular components, the vacuolar membrane (VM) and actin microfilaments (AFs), within the shoot statocyte play important roles in amyloplast dynamics. Flexible VM structures surrounding the amyloplasts seem to allow them to freely sediment toward the bottom of cells. In contrast, long actin cables mediate the saltatory movements of amyloplasts. Thus, amyloplasts in shoot statocytes undergo a dynamic equilibrium of movement, and a proper intracellular environment for statocytes is essential for normal shoot gravitropism. Further analyses to identify the molecular regulators of amyloplast dynamics, including sedimentation, may contribute to an understanding of the gravity sensing mechanism in higher plants.
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http://dx.doi.org/10.3732/ajb.1200315DOI Listing
January 2013

Modulation of the balance between stem cell proliferation and consumption by ERECTA-family genes.

Plant Signal Behav 2012 Nov 18;7(11):1506-8. Epub 2012 Sep 18.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.

Stem cells in the vegetative shoot apical meristem proliferate to produce more stem cells (self-renewal) and are simultaneously consumed to form leaf promordia. Therefore, to keep a stable number of stem cells, regulation of the balance between their proliferation and consumption is important. Recently we reported that stem cell population is increased in mutant plants lacking the entire ERECTA (ER) receptor kinase family. Here we describe that loss of function of the entire ER-family causes a decrease in leaf number in spite of the increase in stem cell population. This suggests that stem cell consumption might be decreased in the mutant, and this could be one of reasons why stem cell population appears to be increased. This situation is in sharp contrast to clv3 mutant, which also shows an increase in stem cell population but does not show a decrease in leaf production. We briefly discuss differences between the er-family mutant and the clv3 mutant.
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http://dx.doi.org/10.4161/psb.22080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548882PMC
November 2012

ERECTA-family receptor kinases regulate stem cell homeostasis via buffering its cytokinin responsiveness in the shoot apical meristem.

Plant Cell Physiol 2013 Mar 9;54(3):343-51. Epub 2012 Aug 9.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0192 Japan.

Shoot apical meristems (SAMs), which are maintained at the tips of stems, are indeterminate structures and sources of stem cells from which all aerial organs are ultimately derived. Although mechanisms that regulate the homeostasis of the stem cells have been extensively investigated, identification of further unknown regulators should provide better understanding of the regulation. Here, we report that members of the Arabidopsis ERECTA (ER) receptor kinase family redundantly play a significant role in the regulation of stem cell homeostasis. In wild-type seedlings, the expression of WUSCHEL (WUS), a central regulator of the stem cell population, is stimulated by cytokinin. Interestingly, however, the SAM morphology and the expression of CLAVATA3 (CLV3), which is expressed in stem cells and therefore serves as a stem cell marker, are relatively stable against cytokinin treatment regardless of increased WUS expression. These findings indicate the presence of a mechanism to buffer stem cell homeostasis against an increase in cytokinin. Mutant seedlings lacking all ER-family members, which are expressed in the SAM, show an increase in the stem cell population and also the up-regulation of a cytokinin-responsive gene in the SAM. In this mutant, WUS expression is stimulated by cytokinin treatment as efficiently as in wild-type plants. However, in contrast to wild-type plants, SAM morphology and CLV3 expression respond drastically to cytokinin treatment, suggesting that the buffering mechanism to maintain stem cell homeostasis against an increase in cytokinin is severely impaired in this mutant. We suggest that the ER family regulates stem cell homeostasis via buffering its cytokinin responsiveness in the SAM.
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http://dx.doi.org/10.1093/pcp/pcs109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589826PMC
March 2013

Interactions of CUP-SHAPED COTYLEDON and SPATULA genes control carpel margin development in Arabidopsis thaliana.

Plant Cell Physiol 2012 Jun 17;53(6):1134-43. Epub 2012 Apr 17.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan.

A characteristic feature of flowering plants is the fusion of carpels, which results in the formation of an enclosed gynoecium. In Arabidopsis thaliana, the gynoecium is formed by the fusion of two carpels along their margins, which also act as a meristematic site for the formation of internal structures such as ovules, the septum and transmitting tract. How gene interactions coordinate the fusion and differentiation of the marginal structures during gynoecium development is largely unknown. It was previously shown that the SPATULA (SPT) gene is required for carpel fusion, whereas overexpression of the CUP-SHAPED COTYLEDON genes CUC1 and CUC2 prevents it. Here we provide evidence that SPT promotes carpel fusion in the apical gynoecium partly through the negative regulation of CUC1 and CUC2 expression. In spt, transcripts of both CUC genes accumulated ectopically, and addition of cuc1 and cuc2 mutations to spt suppressed the split phenotype of carpels specifically along their lateral margins. In the basal gynoecium, on the other hand, all three genes promoted the formation of margin-derived structures, as revealed by the synergistic interactions of spt with each of the cuc mutations. Our results suggest that differential interactions among SPT, CUC1 and CUC2 direct the formation of domain-specific structures of the Arabidopsis gynoecium.
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http://dx.doi.org/10.1093/pcp/pcs057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367164PMC
June 2012
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