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Yasuo Hotta - One of the best experts on this subject based on the ideXlab platform.
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SYNTHESIS OF MESSENGER-LIKE RIBONUCLEIC ACID AND PROTEIN DURING MEIOSIS IN ISOLATED CELLS OF TRILLIUM ERECTUM
2013Co-Authors: Yasuo Hotta, Ph. DAbstract:The synthcsis of RNA and protein by culturcs of isolated Microsporocytes has been demonstratcd. The variation in capacities of such cultures to perform syntheses is a function of meiotic stage and parallels the pattern of changes observed for Microsporocytes in situ. A principal feature of this pattern is the induction of syntheses during pachytenc and diplotcne, stages at which the chromosomcs are partly contracted. By use of Actinomycin D, chloramphenicol, pulsc-labeling with P32-phosphate, and nucleotidc analyses of RNA digests, part of the RNA synthesized has becn shown to corrcspond to messenger RNA. Analysis of reaction rates and the ovcrlappings of protcin and RNA synthcsis indicates that the sprcad of cytological events in Trillium is not purely a function of the low temperature at which it occurs but, presumably, ariscs from a complcment of regulatory devices which govern the periodic onset of reactions within the cells. The main conclusion drawn from the whole of these studies is that the sequence of morphological changes associated with chromosomc contraction and movement during meiosis is accompanied by a set of gene transcriptions. Although comparatively few genes are presumed to be active during meiosis, thc action of such gcnes may be cssential to a translation of some of the information cmbodying the mciotic sequence which has been stored in the gcnome in thc course of evolution
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Characterization of cDNAs induced in meiotic prophase in lily Microsporocytes.
DNA Research, 1994Co-Authors: Toshiyuki Kobayashi, Etsuko Kobayashi, Shusei Sato, Yasuo Hotta, Nobuyuki Miyajima, Ayako Tanaka, Satoshi TabataAbstract:To identify and analyze genes functioning during reproductive cell formation in higher plants, cDNAs harboring the messages induced in meiotic prophase were isolated and characterized. A cDNA library constructed from Microsporocytes in meiotic prophase of Lilium longiflorum was screened with a subtraction probe specific to meiotic prophase. Clones selected were classified into 18 groups by cross hybridization and partial sequencing. Northern blot analysis revealed that the transcripts corresponding to the respective cDNA groups began accumulating at the early stages of meiosis and exhibited clone-specific profiles during meiosis and the spore formation process. The amino acid sequences of the predicted gene products showed similarity with known gene products, e.g. heat shock proteins, serine proteases in Bacillus, and RAD 51 gene product in yeast. Half of the putative gene products had hydrophobic N-terminal regions, suggesting that they may function as signal peptides.
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evidence of meiosis specific regulation of gene expression in lily Microsporocytes
Plant Science, 1993Co-Authors: Satoshi Tabata, Shusei Sato, Yoshinori Watanabe, Masayuki Yamamoto, Yasuo HottaAbstract:Abstract Transfection experiments were performed with DNA constructs bearing the GUS reporter gene and various promoter elements to assess meiosis specificity of expression. Plasmid DNA, pBI221, including the CaM 35S promoter fused to the GUS gene, was expressed in protoplasts derived from lily calli and young anthers but not Microsporocytes. In contrast, plasmid DNAs bearing the meiotic promoter mei2Pro (δ12) and mei2Pro (δ12-s) isolated from Schizosaccharomyces pombe, fused to the GUS gene, were expressed in protoplasts from lily Microsporocytes but not from somatic cells. Furthermore, DNAs with a mutated mei2 promoter fused to the GUS gene showed reduced activity. Transfection of the plasmid DNA, pUC19-recA-mei2, bearing mei2Pro (δ12) in front of the E. coli recA gene, was associated with higher recombinogenic activities assayable in vitro. The induction of GUS in meiocytes by transfection of plasmid DNA was dependent on the presence of mei2, as shown by the reduction of activity when the promoter was mutated and the stage of meiosis. The developmentally regulated expression of a yeast promoter in plant cells demonstrated in this paper may be the first to be reported.
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changes in intracellular camp level and activities of adenylcyclase and phosphodiesterase during meiosis of lily Microsporocytes
Cell Structure and Function, 1992Co-Authors: Shusei Sato, Satoshi Tabata, Yasuo HottaAbstract:In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomyces pombe, reduction of intracellular cyclic adenosine monophosphate (cAMP) is known to trigger the sporulation processes by activating various meiosis specific genes. In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily Microsporocytes. Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.
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isolation of synaptonemal complexes from lily Microsporocytes
Plant Science, 1992Co-Authors: Toshio Ohyama, Yasuo Hotta, Toshiyuki Kobayashi, Yukio Iwaikawa, Satoshi TabataAbstract:Abstract Synaptonemal complexes (SCs) were isolated from Microsporocytes of Lilium. Nuclei prepared from Microsporocytes at pachytene in meiotic prophase were treated with DNase II and SCs released from the nuclei were purified by sedimentation through a density gradient of Nycodenz. The SC fraction thus obtained contained fibrous structures consisting of two parallel filaments as major components. They were ∼ 10 μm in length and 200 nm apart from each other and were considered to be the segments of the lateral elements. According to sodium dodecyl sulfate (SDS)-gel electrophoretic analyses, twenty or more proteins were identified as components of the SC fraction. Among these, five proteins having Mr values of 42, 50, 52 116 and 140 kDa were consistently present in various preparations. Antiserum raised against the isolated SCs recognized several kinds of antigens including the proteins of 42, 50 and 52 kDa, which were likely to be candidates for constituents of SCs.
Satoshi Tabata - One of the best experts on this subject based on the ideXlab platform.
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Characterization of cDNAs induced in meiotic prophase in lily Microsporocytes.
DNA Research, 1994Co-Authors: Toshiyuki Kobayashi, Etsuko Kobayashi, Shusei Sato, Yasuo Hotta, Nobuyuki Miyajima, Ayako Tanaka, Satoshi TabataAbstract:To identify and analyze genes functioning during reproductive cell formation in higher plants, cDNAs harboring the messages induced in meiotic prophase were isolated and characterized. A cDNA library constructed from Microsporocytes in meiotic prophase of Lilium longiflorum was screened with a subtraction probe specific to meiotic prophase. Clones selected were classified into 18 groups by cross hybridization and partial sequencing. Northern blot analysis revealed that the transcripts corresponding to the respective cDNA groups began accumulating at the early stages of meiosis and exhibited clone-specific profiles during meiosis and the spore formation process. The amino acid sequences of the predicted gene products showed similarity with known gene products, e.g. heat shock proteins, serine proteases in Bacillus, and RAD 51 gene product in yeast. Half of the putative gene products had hydrophobic N-terminal regions, suggesting that they may function as signal peptides.
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evidence of meiosis specific regulation of gene expression in lily Microsporocytes
Plant Science, 1993Co-Authors: Satoshi Tabata, Shusei Sato, Yoshinori Watanabe, Masayuki Yamamoto, Yasuo HottaAbstract:Abstract Transfection experiments were performed with DNA constructs bearing the GUS reporter gene and various promoter elements to assess meiosis specificity of expression. Plasmid DNA, pBI221, including the CaM 35S promoter fused to the GUS gene, was expressed in protoplasts derived from lily calli and young anthers but not Microsporocytes. In contrast, plasmid DNAs bearing the meiotic promoter mei2Pro (δ12) and mei2Pro (δ12-s) isolated from Schizosaccharomyces pombe, fused to the GUS gene, were expressed in protoplasts from lily Microsporocytes but not from somatic cells. Furthermore, DNAs with a mutated mei2 promoter fused to the GUS gene showed reduced activity. Transfection of the plasmid DNA, pUC19-recA-mei2, bearing mei2Pro (δ12) in front of the E. coli recA gene, was associated with higher recombinogenic activities assayable in vitro. The induction of GUS in meiocytes by transfection of plasmid DNA was dependent on the presence of mei2, as shown by the reduction of activity when the promoter was mutated and the stage of meiosis. The developmentally regulated expression of a yeast promoter in plant cells demonstrated in this paper may be the first to be reported.
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changes in intracellular camp level and activities of adenylcyclase and phosphodiesterase during meiosis of lily Microsporocytes
Cell Structure and Function, 1992Co-Authors: Shusei Sato, Satoshi Tabata, Yasuo HottaAbstract:In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomyces pombe, reduction of intracellular cyclic adenosine monophosphate (cAMP) is known to trigger the sporulation processes by activating various meiosis specific genes. In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily Microsporocytes. Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.
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isolation of synaptonemal complexes from lily Microsporocytes
Plant Science, 1992Co-Authors: Toshio Ohyama, Yasuo Hotta, Toshiyuki Kobayashi, Yukio Iwaikawa, Satoshi TabataAbstract:Abstract Synaptonemal complexes (SCs) were isolated from Microsporocytes of Lilium. Nuclei prepared from Microsporocytes at pachytene in meiotic prophase were treated with DNase II and SCs released from the nuclei were purified by sedimentation through a density gradient of Nycodenz. The SC fraction thus obtained contained fibrous structures consisting of two parallel filaments as major components. They were ∼ 10 μm in length and 200 nm apart from each other and were considered to be the segments of the lateral elements. According to sodium dodecyl sulfate (SDS)-gel electrophoretic analyses, twenty or more proteins were identified as components of the SC fraction. Among these, five proteins having Mr values of 42, 50, 52 116 and 140 kDa were consistently present in various preparations. Antiserum raised against the isolated SCs recognized several kinds of antigens including the proteins of 42, 50 and 52 kDa, which were likely to be candidates for constituents of SCs.
Dazhong Zhao - One of the best experts on this subject based on the ideXlab platform.
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control of anther cell differentiation by the small protein ligand tpd1 and its receptor ems1 in arabidopsis
PLOS Genetics, 2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive Microsporocytes (pollen mother cells) and surrounding somatic cells. The Microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS Microsporocytes1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of Microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction.
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Control of Anther Cell Differentiation by the Small Protein Ligand TPD1 and Its Receptor EMS1 in Arabidopsis.
Public Library of Science (PLoS), 2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive Microsporocytes (pollen mother cells) and surrounding somatic cells. The Microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS Microsporocytes1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of Microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction
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Genetic ablation of Microsporocytes shows the interdependence of tapetal cell and microsporocyte differentiation.
2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:(A) A wild-type anther lobe at stage 5 showing four layers of anther wall cells (indicated by red dots, the same hereinafter) and Microsporocytes. (B-E) SDS:SDS-BANASE anther lobes at stage 5, which we divided into three classes. Class I: four somatic cell layers, including one organized single-cell layer that surrounds the Microsporocytes and is made of cells that are morphologically similar to tapetal cells (B). Class II: four somatic cell layers, including a monolayer of vacuolated tapetal-like cells (C) and three somatic cell layers that contains delaminated vacuolated tapetal-like cells (D). Degenerating Microsporocytes are observed in Class-II anthers. Class III: three somatic cell layers and excess Microsporocytes (E). Among 60 T1 plants analyzed by semi-thin section, 16.7% (10/60) were Class I, 70.0% (42/60) were Class II, and 13.3% (8/60) were Class III. (F-J) In situ hybridization results showing that the expression of the tapetal cell marker gene, A9 at stage 5 was strong in the tapetum of the wild-type anther (F), but was progressively decreased in tapetal-like cells from Class-I (G) and Class-II (H, I) SDS:SDS-BANASE anthers. No A9 expression was detected in the Class-III SDS:SDS-BANASE anther (J). (K-O) In situ hybridization results showing the expression of the microsporocyte marker gene, SDS, in anthers. In wild-type anthers, SDS expression was weak at stage 4 in precursors of Microsporocytes (K), but strong at stage 5 in Microsporocytes (L). SDS was weakly expressed in the Microsporocytes of SDS:SDS-BANASE anthers (M-O). The SDS expression domain was relatively expanded in SDS:SDS-BANASE Class-II and -III anthers (N, O). E, epidermis; En, endothecium; M, microsporocyte; ML, middle layer; PM, precursor of microsporocyte; T, tapetal cell; and TL, tapetal-like cell. Scale bars, 10 μm. (P, Q) We used qRT-PCR to examine expression levels of A9 and BARNASE in anthers from three representative transgenic plants of each class. From Class I to Class III anthers, the expression of A9 progressively decreased (P), while that of BARNASE increased (Q).
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Localization of TPD1 and EMS1 in anthers.
2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:(A-T) Confocal images showing merges of red chlorophyll autofluorescence and green GFP signals, with the exceptions of (G) and (H). (A, B) In TPD1:mGFP5er stage-4 (A) and stage-5 (B) anthers, the TPD1 promoter was active only in precursors of Microsporocytes and Microsporocytes, respectively. (C, D) In the TPD1:TPD1sp-GFP-ΔTPD1/tpd1 stage-4 anther, TPD1 proteins were detected in precursors of Microsporocytes (D, high magnification of C). (E, F) In the TPD1:TPD1sp-GFP-ΔTPD1/tpd1 stage-5 anther, TPD proteins were mainly localized in Microsporocytes, but were also detected at the surface of tapetal cells (F, high magnification of E). (G, H) TPD1 proteins were localized in vesicle-like compartments of Microsporocytes isolated from TPD1:TPD1sp-GFP-ΔTPD1/tpd1 stage-5 anthers (H, confocal image merged with DIC-viewed Microsporocytes). (I, J) In the TPD1:TPD1sp-GFP-ΔTPD1/ems1 stage-5 anther, the TPD1 localization domain was expanded and TPD1 proteins were evenly distributed in Microsporocytes as these anthers lacked tapetal cells (J, high magnification of I). (K, L) In TPD1:GFP-ΔTPD1 (K) and TPD1:TPD1sp-GFP-ΔTPD1K135G R136G (L) stage-5 anthers, TPD1 proteins were restricted to Microsporocytes, regardless of the presence of EMS1. (M, N) In the EMS1:mGFP5er stage-4 anther, the EMS1 promoter was active in outer secondary parietal cells (OSPC) and inner secondary parietal cells (ISPC) (N, high magnification of M). (O, P) In the early EMS1:mGFP5er stage-5 anther, the EMS1 promoter was active in the middle layer (ML) and precursors of tapetal cells (PT) (P, high magnification of O). (Q, R) In the EMS1:mGFP5er stage-5 anther, EMS1 promoter activity was only detected in tapetal cells (T) (R, high magnification of Q). (S, T) In the EMS1:EMS1-3xGFP/ems1 stage-5 anther, EMS1 proteins were only observed at surfaces of tapetal cells (T shows a higher magnification of S). (U, V) EM-immunolabeling results showing TPD1 (U) and EMS1 (V) proteins at the plasma membrane of precursors of tapetal cells from TPD1:TPD1sp-GFP-ΔTPD1/tpd1 and EMS1:EMS1-3xGFP/ems1 early stage-5 anthers, respectively. For each GFP fusion gene, at least 15 independent T2 plants were observed. Similar GFP signals were observed from >90% tested plants. The anther stage was determined by FM4-64 staining (See S5 Fig) after GFP images were acquired. ISPC, inner secondary parietal cell; M, microsporocyte; ML, middle layer; OSPC, outer secondary parietal cell; PM, precursor of microsporocyte; PT, precursor of tapetal cell; S, stage; and T, tapetal cell. (A-C, E, I, K, L, M, O, Q, S) Scale bars, 50 μm. (D, F, J, N, P, R, T) Scale bars, 20 μm. (G, H) Scale bars, 10 μm. (U, V) Scale bars, 0.2 μm.
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Missorting of TDP1 demonstrates that microsporocyte-derived TPD1 mediates the acquisition of tapetal cell fate.
2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:(A) Schematic diagrams showing the structures of the TPD1:TPD1, TPD1:TPD1-ctVSS, and TPD1:TPD1-ctVSS-GG constructs. Red bar: the TPD1 putative signal peptide, Cyan bar: the non-conserved N-terminal region, Blue bar: the conserved C-terminal domain, Orange bar, the C-terminal vacuole sorting signal (ctVSS), and “GG”: two glycines added to ctVSS. (B-D) Primary inflorescences showing normal fertilities (indicated by long siliques) were obtained from TPD1:TPD1/tpd1 (B) and TPD1:TPD1-ctVSS-GG/tpd1 (D) plants, whereas TPD1:TPD1-ctVSS/tpd1 plants were sterile (as indicated by short siliques) (C). Scale bars, 1 cm. (E) Complementation rates (%) of TPD1:TPD1/tpd1 (n = 40), TPD1:TPD1-ctVSS/tpd1 (n = 60), and TPD1:TPD1-ctVSS-GG/tpd1 (n = 56) plants. (F) A stage-5 anther lobe from a TPD1:TPD1/tpd1 plant showing normal anther cell differentiation. (G-I) Stage-5 anther lobes from TPD1:TPD1-ctVSS/tpd1 plants showing defective anther cell differentiation: a monolayer of vacuolated tapetal-like cells and degenerating Microsporocytes (G), delaminated vacuolated tapetal-like cells and degenerating Microsporocytes (H), and a lack of tapetal cells coupled with the presence of excess Microsporocytes, which is similar to the tpd1 phenotype (I). Thirty sterile TPD1:TPD1-ctVSS/tpd1 plants were subjected to semi-thin sectioning. Among them, most (21/30) exhibited the anther phenotype shown in (G), five exhibited that shown in (H), and four exhibited that shown in (I). E, epidermis; En, endothecium; M, microsporocyte; ML, middle layer; T, tapetal cell; and TL, tapetal-like cells. Scale bars, 10 μm.
Jian Huang - One of the best experts on this subject based on the ideXlab platform.
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carbonic anhydrases function in anther cell differentiation downstream of the receptor like kinase ems1
The Plant Cell, 2017Co-Authors: Jian Huang, Zhiyong Li, Gabriel Biener, Erhui Xiong, Shikha Malik, Nathan Eaton, Catherine Z Zhao, Valerica Raicu, Hongzhi Kong, Dazhong Dave ZhaoAbstract:Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS Microsporocytes1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive Microsporocytes during early anther development in Arabidopsis thaliana. Here, we report the identification of β-carbonic anhydrases (βCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with βCA proteins. Loss-of-function of βCA genes caused defective tapetal cell differentiation, while overexpression of βCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates βCA1 at four sites, resulting in increased βCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of βCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. βCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of βCA in controlling cell differentiation and provide insights into the post-translational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation.
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control of anther cell differentiation by the small protein ligand tpd1 and its receptor ems1 in arabidopsis
PLOS Genetics, 2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive Microsporocytes (pollen mother cells) and surrounding somatic cells. The Microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS Microsporocytes1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of Microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction.
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Control of Anther Cell Differentiation by the Small Protein Ligand TPD1 and Its Receptor EMS1 in Arabidopsis.
Public Library of Science (PLoS), 2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive Microsporocytes (pollen mother cells) and surrounding somatic cells. The Microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS Microsporocytes1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of Microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction
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Genetic ablation of Microsporocytes shows the interdependence of tapetal cell and microsporocyte differentiation.
2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:(A) A wild-type anther lobe at stage 5 showing four layers of anther wall cells (indicated by red dots, the same hereinafter) and Microsporocytes. (B-E) SDS:SDS-BANASE anther lobes at stage 5, which we divided into three classes. Class I: four somatic cell layers, including one organized single-cell layer that surrounds the Microsporocytes and is made of cells that are morphologically similar to tapetal cells (B). Class II: four somatic cell layers, including a monolayer of vacuolated tapetal-like cells (C) and three somatic cell layers that contains delaminated vacuolated tapetal-like cells (D). Degenerating Microsporocytes are observed in Class-II anthers. Class III: three somatic cell layers and excess Microsporocytes (E). Among 60 T1 plants analyzed by semi-thin section, 16.7% (10/60) were Class I, 70.0% (42/60) were Class II, and 13.3% (8/60) were Class III. (F-J) In situ hybridization results showing that the expression of the tapetal cell marker gene, A9 at stage 5 was strong in the tapetum of the wild-type anther (F), but was progressively decreased in tapetal-like cells from Class-I (G) and Class-II (H, I) SDS:SDS-BANASE anthers. No A9 expression was detected in the Class-III SDS:SDS-BANASE anther (J). (K-O) In situ hybridization results showing the expression of the microsporocyte marker gene, SDS, in anthers. In wild-type anthers, SDS expression was weak at stage 4 in precursors of Microsporocytes (K), but strong at stage 5 in Microsporocytes (L). SDS was weakly expressed in the Microsporocytes of SDS:SDS-BANASE anthers (M-O). The SDS expression domain was relatively expanded in SDS:SDS-BANASE Class-II and -III anthers (N, O). E, epidermis; En, endothecium; M, microsporocyte; ML, middle layer; PM, precursor of microsporocyte; T, tapetal cell; and TL, tapetal-like cell. Scale bars, 10 μm. (P, Q) We used qRT-PCR to examine expression levels of A9 and BARNASE in anthers from three representative transgenic plants of each class. From Class I to Class III anthers, the expression of A9 progressively decreased (P), while that of BARNASE increased (Q).
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Localization of TPD1 and EMS1 in anthers.
2016Co-Authors: Jian Huang, Tianyu Zhang, Lisa Linstroth, Zachary Tillman, Marisa S Otegui, Heather A Owen, Dazhong ZhaoAbstract:(A-T) Confocal images showing merges of red chlorophyll autofluorescence and green GFP signals, with the exceptions of (G) and (H). (A, B) In TPD1:mGFP5er stage-4 (A) and stage-5 (B) anthers, the TPD1 promoter was active only in precursors of Microsporocytes and Microsporocytes, respectively. (C, D) In the TPD1:TPD1sp-GFP-ΔTPD1/tpd1 stage-4 anther, TPD1 proteins were detected in precursors of Microsporocytes (D, high magnification of C). (E, F) In the TPD1:TPD1sp-GFP-ΔTPD1/tpd1 stage-5 anther, TPD proteins were mainly localized in Microsporocytes, but were also detected at the surface of tapetal cells (F, high magnification of E). (G, H) TPD1 proteins were localized in vesicle-like compartments of Microsporocytes isolated from TPD1:TPD1sp-GFP-ΔTPD1/tpd1 stage-5 anthers (H, confocal image merged with DIC-viewed Microsporocytes). (I, J) In the TPD1:TPD1sp-GFP-ΔTPD1/ems1 stage-5 anther, the TPD1 localization domain was expanded and TPD1 proteins were evenly distributed in Microsporocytes as these anthers lacked tapetal cells (J, high magnification of I). (K, L) In TPD1:GFP-ΔTPD1 (K) and TPD1:TPD1sp-GFP-ΔTPD1K135G R136G (L) stage-5 anthers, TPD1 proteins were restricted to Microsporocytes, regardless of the presence of EMS1. (M, N) In the EMS1:mGFP5er stage-4 anther, the EMS1 promoter was active in outer secondary parietal cells (OSPC) and inner secondary parietal cells (ISPC) (N, high magnification of M). (O, P) In the early EMS1:mGFP5er stage-5 anther, the EMS1 promoter was active in the middle layer (ML) and precursors of tapetal cells (PT) (P, high magnification of O). (Q, R) In the EMS1:mGFP5er stage-5 anther, EMS1 promoter activity was only detected in tapetal cells (T) (R, high magnification of Q). (S, T) In the EMS1:EMS1-3xGFP/ems1 stage-5 anther, EMS1 proteins were only observed at surfaces of tapetal cells (T shows a higher magnification of S). (U, V) EM-immunolabeling results showing TPD1 (U) and EMS1 (V) proteins at the plasma membrane of precursors of tapetal cells from TPD1:TPD1sp-GFP-ΔTPD1/tpd1 and EMS1:EMS1-3xGFP/ems1 early stage-5 anthers, respectively. For each GFP fusion gene, at least 15 independent T2 plants were observed. Similar GFP signals were observed from >90% tested plants. The anther stage was determined by FM4-64 staining (See S5 Fig) after GFP images were acquired. ISPC, inner secondary parietal cell; M, microsporocyte; ML, middle layer; OSPC, outer secondary parietal cell; PM, precursor of microsporocyte; PT, precursor of tapetal cell; S, stage; and T, tapetal cell. (A-C, E, I, K, L, M, O, Q, S) Scale bars, 50 μm. (D, F, J, N, P, R, T) Scale bars, 20 μm. (G, H) Scale bars, 10 μm. (U, V) Scale bars, 0.2 μm.
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Attachment of kinetochores to spindle microtubules during meiosis I of Lilium Microsporocytes
Chromosome Research, 1999Co-Authors: T Suzuki, Isao TanakaAbstract:Kinetochores and microtubules were visualized simultaneously during spindle formation at the first meiotic division in Microsporocytes of Lilium longiflorum (2n = 24) under a confocal laser-scanning microscope, after immunofluorescence staining with centromere-recognizing antiserum and tubulin-specific antibody. During early prometaphase I, each kinetochore of bivalent chromosomes appeared to be an amorphous flat structure upon its initial attachment to microtubules. It became compact and spherical with the development of the spindle. From late prometaphase I, when the bipolar spindle was nearly complete, each kinetochore resembled a double disk that was suggestive of a pair of sister kinetochores and the homologous kinetochores were oriented towards opposite poles. Thus, the bipolar spindle at metaphase I included 12 bivalent chromosomes with a total of four kinetochores each. At anaphase I, the sister kinetochores moved to the same spindle pole as a paired unit. In Microsporocytes arrested at prometaphase I by colchicine treatment, the sister kinetochores also came to be distinguishable. These results suggest that the change of kinetochore structure during meiosis I may be under chromosomal control but be somewhat associated with its attachment to spindle microtubules.