scholarly journals Carbon Metabolism in Developing Soybean Root Nodules: The Role of Carbonic Anhydrase

2000 ◽  
Vol 13 (1) ◽  
pp. 14-22 ◽  
Author(s):  
Nektarios Kavroulakis ◽  
Emanouil Flemetakis ◽  
Georgios Aivalakis ◽  
Panagiotis Katinakis
Keyword(s):  
In Situ Hybridization ◽  
Carbonic Anhydrase ◽  
Root Nodules ◽  
Cell Types ◽  
Nodule Development ◽  
Vascular Bundles ◽  
Transcript Accumulation ◽  
Cortical Cells ◽  
Gene Transcripts

A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development.

scholarly journals Aspartate Aminotransferase in Alfalfa Nodules: Localization of mRNA During Effective and Ineffective Nodule Development and Promoter Analysis

1999 ◽  
Vol 12 (4) ◽  
pp. 263-274 ◽  
Author(s):  
Hirofumi Yoshioka ◽  
Robert G. Gregerson ◽  
Deborah A. Samac ◽  
Kim C. M. Hoevens ◽  
Gian Trepp ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Root Nodules ◽  
Critical Role ◽  
Nodule Development ◽  
Vascular Bundles ◽  
Fixed Nitrogen ◽  
Transcript Accumulation ◽  
Specific Expression ◽  
Promoter Deletion Analysis

Aspartate aminotransferase (AAT) plays a critical role in the assimilation of symbiotically fixed nitrogen into aspartate and asparagine in legume root nodules. The enzyme occurs as a cytosolic form (AAT1) and a plastid form (AAT2) in alfalfa nodules. To elucidate the functional role of each isozyme in root nodule metabolism further, in situ hybridization was used to determine the pattern of transcript accumulation from the two genes. AAT2 transcripts were localized to infected cells throughout the symbiotic zone of effective alfalfa nodules; however, expression was reduced in ineffective nodules. The AAT1 gene was expressed in the uninfected cells of the invasion zone and symbiotic zone, the nodule parenchyma, and nodule vascular bundles of both effective and ineffective nodules. The AAT1 and AAT2 promoters were evaluated in transgenic alfalfa plants containing promoter β-glucuronidase (GUS) gene fusions. Histochemical staining patterns agreed with results from in situ localization. The distribution pattern of gene transcripts suggests that AAT1 has a role in maintenance of the O2 diffusion barrier in nodules and that AAT2 plays a major role in assimilation of recently fixed nitrogen. Promoter deletion analysis of the AAT2 promoter revealed that nodule-specific expression was retained in a promoter fragment of 300 bp.

scholarly journals Detection of estrogen receptor alpha, carbonic anhydrase II and tartrate-resistant acid phosphatase mRNAs in putative mononuclear osteoclast precursor cells of neonatal rats by fluorescence in situ hybridization

1998 ◽  
Vol 20 (2) ◽  
pp. 211-219 ◽  
Author(s):  
WH Huang ◽  
AT Lau ◽  
LL Daniels ◽  
H Fujii ◽  
U Seydel ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
In Situ Hybridization ◽  
Acid Phosphatase ◽  
Carbonic Anhydrase ◽  
Precursor Cells ◽  
Osteoclast Precursor ◽  
Carbonic Anhydrase Ii ◽  
Tartrate Resistant Acid Phosphatase ◽  
Gene Transcripts

Increasing evidence suggests that estrogen deficiency in women promotes the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and local factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II), characterizes osteoclast differentiation. To evaluate the possibility that estrogen may have a direct effect on osteoclast precursor cells, we investigated the mRNA levels of estrogen receptor a (ERa), TRACP and CA II genes in neonatal rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display strongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions similar to those observed in osteoclasts. Both CA II and TRACP mRNAs were detected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To determine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in their cytoplasm. In contrast, only a few multinuclear osteoclasts in the bone imprints possessed ER gene transcripts. Interestingly, the levels of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybridization resulted in a significant loss of signal in these cells. The results of these studies suggest that estrogen may have a direct role in modulating the recruitment of osteoclast precursor cells during osteoclastogenesis.

scholarly journals 087 Localization of ENOD2 Transcript Accumulation in Indeterminant Nodules of Maackia amurensis Rupr. & Maxim. (Amur Maackia)

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 456C-456
Author(s):  
Harry T. Horner ◽  
David J. Hannapel ◽  
William R. Graves ◽  
Carol M. Foster ◽  
David J. Hannapel ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Periodic Acid ◽  
Nodule Development ◽  
Coding Region ◽  
Transcript Accumulation ◽  
Mrna Accumulation ◽  
Peripheral Tissues ◽  
Maackia Amurensis ◽  
Early Nodulin

Early nodulin genes, such as ENOD2, play a role in the first stages of nodulation. Although ENOD2 is conserved among nodulating legumes studied to date, its occurrence and activity have not been studied among woody legumes such as Maackia amurensis Rupr. & Maxim. Our objective was to localize MaENOD2 transcripts during nodule development and describe the anatomy of nodules formed on the roots of M. amurensis in relation to ENOD2 mRNA accumulation. Nodules (<1 mm, 1-2 mm, >2 mm in diameter, and mature) were prepared for light microscopy, sectioned, and stained with safranin and fast green for structural contrast or with the periodic acid Schiff's reaction for starch. The location of ENOD2 transcripts was determined by using in situ hybridization with DIG-labeled sense and antisense RNAs transcribed from a 602-bp fragment of the coding region of MaENOD2. Mature nodules from M. amurensis possessed peripheral tissues, a distal meristem, and a central infected region characteristic of indeterminant development. In situ hybridization showed that MaENOD2 transcripts accumulated in the distribution layer and uninfected cells of the central symbiotic region. Amyloplasts that contained starch grains were identified in these tissues and in the inner parenchyma of the nodule. Throughout nodule development, transcripts were restricted to areas with high levels of stored starch that surrounded cells actively fixing N2. Our results suggest that ENOD2 in M. amurensis may be a cell wall component of tissues that regulate nutrient flow to and from sinks, such as symbiotic regions of a nodule. These data may lead to a better understanding of the role of the ENOD2 gene family during nodulation.

Pituitary adenoma producing growth hormone and adrenocorticotropin: a histological, immunocytochemical, electron microscopic, and in situ hybridization study

1998 ◽  
Vol 88 (6) ◽  
pp. 1111-1115 ◽  
Author(s):  
Kalman Kovacs ◽  
Eva Horvath ◽  
Lucia Stefaneanu ◽  
Juan Bilbao ◽  
William Singer ◽  
...  
Keyword(s):  
Growth Hormone ◽  
In Situ Hybridization ◽  
Pituitary Adenoma ◽  
Immunoelectron Microscopy ◽  
Secretory Granules ◽  
Cell Types ◽  
Content Type ◽  
Separate Cell ◽  
Fine Print

✓ The authors report on the morphological features of a pituitary adenoma that produced growth hormone (GH) and adrenocorticotropic hormone (ACTH). This hormone combination produced by a single adenoma is extremely rare; a review of the available literature showed that only one previous case has been published. The tumor, which was removed from a 62-year-old man with acromegaly, was studied by histological and immunocytochemical analyses, transmission electron microscopy, immunoelectron microscopy, and in situ hybridization. When the authors used light microscopy, the tumor appeared to be a bimorphous mixed pituitary adenoma composed of two separate cell types: one cell population synthesized GH and the other ACTH. The cytogenesis of pituitary adenomas that produce more than one hormone is obscure. It may be that two separate cells—one somatotroph and one corticotroph—transformed into neoplastic cells, or that the adenoma arose in a common stem cell that differentiated into two separate cell types. In this case immunoelectron microscopy conclusively demonstrated ACTH in the secretory granules of several somatotrophs. This was associated with a change in the morphological characteristics of secretory granules. Thus it is possible that the tumor was originally a somatotropic adenoma that began to produce ACTH as a result of mutations that occurred during tumor progression.

scholarly journals Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ

1999 ◽  
Vol 65 (4) ◽  
pp. 1746-1752 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Microbial Communities ◽  
Fluorescent In Situ Hybridization ◽  
Amino Acid Uptake ◽  
Cell Types ◽  
Black Box ◽  
Acid Uptake ◽  
Substrate Uptake

ABSTRACT We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria(α-Proteobacteria) and of the Cytophaga-Flavobacteriumgroup obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (∼60% of the universal probe-labeled cells and ∼50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.

scholarly journals PvRACK1 Loss-of-Function Impairs Cell Expansion and Morphogenesis in Phaseolus vulgaris L. Root Nodules

2011 ◽  
Vol 24 (7) ◽  
pp. 819-826 ◽  
Author(s):  
Tania Islas-Flores ◽  
Gabriel Guillén ◽  
Xóchitl Alvarado-Affantranger ◽  
Miguel Lara-Flores ◽  
Federico Sánchez ◽  
...  
Keyword(s):  
Phaseolus Vulgaris ◽  
Cell Expansion ◽  
Root Nodules ◽  
Microscopic Analysis ◽  
Nodule Development ◽  
Loss Of Function ◽  
Rhizobium Tropici ◽  
Transcript Accumulation ◽  
Transgenic Roots ◽  
Plant Signal Transduction

Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar β-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.

scholarly journals Expression Analysis of PIN Genes in Root Tips and Nodules of Lotus japonicus

2019 ◽  
Vol 20 (2) ◽  
pp. 235 ◽  
Author(s):  
Izabela Sańko-Sawczenko ◽  
Dominika Dmitruk ◽  
Barbara Łotocka ◽  
Elżbieta Różańska ◽  
Weronika Czarnocka
Keyword(s):  
Root Nodule ◽  
Root Nodules ◽  
Quantitative Polymerase Chain Reaction ◽  
Lotus Japonicus ◽  
Nodule Development ◽  
Vascular Bundles ◽  
Root Tips ◽  
Gus Reporter ◽  
Gene Assay ◽  
Development And Differentiation

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical β-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root’s stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation.

scholarly journals Methods to Enhance Signal Using Isotopic In Situ Hybridization

2002 ◽  
Vol 50 (8) ◽  
pp. 1031-1037 ◽  
Author(s):  
Betty Ky ◽  
Paul J. Shughrue
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cell Types ◽  
Oxytocin Receptor ◽  
Specific Cell ◽  
Rat Hypothalamus ◽  
Low Levels ◽  
Tuberoinfundibular Peptide ◽  
Cryostat Sections

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

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