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

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.1.14 ◽

2000 ◽

Vol 13 (1) ◽

pp. 14-22 ◽

Cited By ~ 31

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.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-11-10-0261 ◽

2011 ◽

Vol 24 (7) ◽

pp. 819-826 ◽

Cited By ~ 20

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20020235 ◽

2019 ◽

Vol 20 (2) ◽

pp. 235 ◽

Cited By ~ 4

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.

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

HortScience ◽

10.21273/hortsci.34.3.456c ◽

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.

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Glutathione Plays a Fundamental Role in Growth and Symbiotic Capacity of Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.187.1.168-174.2005 ◽

2005 ◽

Vol 187 (1) ◽

pp. 168-174 ◽

Cited By ~ 63

Author(s):

Judith Harrison ◽

Alexandre Jamet ◽

Cecilia I. Muglia ◽

Ghislaine Van de Sype ◽

O. Mario Aguilar ◽

...

Keyword(s):

Sinorhizobium Meliloti ◽

Root Nodules ◽

Critical Role ◽

Nodule Development ◽

Second Step ◽

Symbiotic Relationship ◽

Wild Type ◽

Free Living ◽

Mutant Strains ◽

Fixation Capacity

ABSTRACT Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We have examined the importance of glutathione (GSH) during free-living growth and symbiosis of Sinorhizobium meliloti. An S. meliloti mutant strain (SmgshA) which is unable to synthesize GSH due to a gene disruption in gshA, encoding the enzyme for the first step in the biosynthesis of GSH, was unable to grow under nonstress conditions, precluding any nodulation. In contrast, an S. meliloti strain (SmgshB) with gshB, encoding the enzyme involved in the second step in GSH synthesis, deleted was able to grow, indicating that γ-glutamylcysteine, the dipeptide intermediate, can partially substitute for GSH. However, the SmgshB strain showed a delayed-nodulation phenotype coupled to a 75% reduction in the nitrogen fixation capacity. This phenotype was linked to abnormal nodule development. Both the SmgshA and SmgshB mutant strains exhibited higher catalase activity than the wild-type S. meliloti strain, suggesting that both mutant strains are under oxidative stress. Taken together, these results show that GSH plays a critical role in the growth of S. meliloti and during its interaction with the plant partner.

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Genome-wide identification of cyclin-dependent kinase (CDK) genes affecting adipocyte differentiation in cattle

BMC Genomics ◽

10.1186/s12864-021-07653-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Cuili Pan ◽

Zhaoxiong Lei ◽

Shuzhe Wang ◽

Xingping Wang ◽

Dawei Wei ◽

...

Keyword(s):

Gene Family ◽

Expression Analysis ◽

Adipocyte Differentiation ◽

Bos Taurus ◽

Adipogenic Differentiation ◽

Critical Role ◽

Bos Indicus ◽

Specific Expression ◽

Genome Wide ◽

A Genome

Abstract Background Cyclin-dependent kinases (CDKs) are protein kinases regulating important cellular processes such as cell cycle and transcription. Many CDK genes also play a critical role during adipogenic differentiation, but the role of CDK gene family in regulating bovine adipocyte differentiation has not been studied. Therefore, the present study aims to characterize the CDK gene family in bovine and study their expression pattern during adipocyte differentiation. Results We performed a genome-wide analysis and identified a number of CDK genes in several bovine species. The CDK genes were classified into 8 subfamilies through phylogenetic analysis. We found that 25 bovine CDK genes were distributed in 16 different chromosomes. Collinearity analysis revealed that the CDK gene family in Bos taurus is homologous with Bos indicus, Hybrid-Bos taurus, Hybrid Bos indicus, Bos grunniens and Bubalus bubalis. Several CDK genes had higher expression levels in preadipocytes than in differentiated adipocytes, as shown by RNA-seq analysis and qPCR, suggesting a role in the growth of emerging lipid droplets. Conclusion In this research, 185 CDK genes were identified and grouped into eight distinct clades in Bovidae, showing extensively homology. Global expression analysis of different bovine tissues and specific expression analysis during adipocytes differentiation revealed CDK4, CDK7, CDK8, CDK9 and CDK14 may be involved in bovine adipocyte differentiation. The results provide a basis for further study to determine the roles of CDK gene family in regulating adipocyte differentiation, which is beneficial for beef quality improvement.

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Molecular modulation of calcium oxalate crystallization

AJP Renal Physiology ◽

10.1152/ajprenal.00136.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. F1123-F1132 ◽

Cited By ~ 57

Author(s):

James J. De Yoreo ◽

S. Roger Qiu ◽

John R. Hoyer

Keyword(s):

Molecular Modeling ◽

Calcium Oxalate ◽

Stone Formation ◽

Energy Levels ◽

Critical Role ◽

Specific Interactions ◽

Crystal Surfaces ◽

Site Specific

Calcium oxalate monohydrate (COM) is the primary constituent of the majority of renal stones. Osteopontin (OPN), an aspartic acid-rich urinary protein, and citrate, a much smaller molecule, are potent inhibitors of COM crystallization at levels present in normal urine. Current concepts of the role of site-specific interactions in crystallization derived from studies of biomineralization are reviewed to provide a context for understanding modulation of COM growth at a molecular level. Results from in situ atomic force microscopy (AFM) analyses of the effects of citrate and OPN on growth verified the critical role of site-specific interactions between these growth modulators and individual steps on COM crystal surfaces. Molecular modeling investigations of interactions of citrate with steps and faces on COM crystal surfaces provided links between the stereochemistry of interaction and the binding energy levels that underlie mechanisms of growth modification and changes in overall crystal morphology. The combination of in situ AFM and molecular modeling provides new knowledge that will aid rationale design of therapeutic agents for inhibition of stone formation.

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Localization of type 1 17beta-hydroxysteroid dehydrogenase mRNA and protein in syncytiotrophoblasts and invasive cytotrophoblasts in the human term villi

Journal of Endocrinology ◽

10.1677/joe.0.1650217 ◽

2000 ◽

Vol 165 (2) ◽

pp. 217-222 ◽

Cited By ~ 18

Author(s):

M Bonenfant ◽

PR Provost ◽

R Drolet ◽

Y Tremblay

Keyword(s):

In Situ Hybridization ◽

Hydroxysteroid Dehydrogenase ◽

Binding Activity ◽

Placental Lactogen ◽

Specific Expression ◽

P450 Aromatase ◽

Chain Cleavage ◽

Extravillous Cytotrophoblasts

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the synthesis of sex steroids. The hallmark of this family of enzymes is the interconversion, through their oxydoreductive reactivity at position C17, of 17-keto- and 17beta-hydroxy-steroids. Because this reaction essentially transforms steroids having low binding activity for the steroid receptor to their more potent 17beta-hydroxysteroids isoforms, it is crucial to the control of the physiological activities of both estrogens and androgens. The human placenta produces large amounts of progesterone and estrogens throughout pregnancy. The placental type 1 17beta-HSD enzyme (E17beta-HSD) catalyzes the reduction of the low activity estrogen, estrone, into the potent estrogen, estradiol. We studied the cell-specific expression of type 1 17beta-HSD in human term placental villous tissue by combining in situ hybridization to localize type 1 17beta-HSD mRNA with immunohistochemistry using an antibody against human placental lactogen, a trophoblast marker. Immunolocalization of E17beta-HSD was also performed. To ascertain whether other steroidogenic enzymes are present in the same cell type, cytochrome P450 cholesterol side-chain cleavage (P450scc), P450 aromatase, and type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were also localized by immunostaining. Our results showed that the syncytium is the major steroidogenic unit of the fetal term villi. In fact, type 1 17beta-HSD mRNA and protein, as well as P450scc, P450 aromatase, and 3beta-HSD immunoreactivities were found in these cells. In addition, our results revealed undoubtedly that extravillous cytotrophoblasts (CTBs), e.g. those from which cell columns of anchoring villous originate, also express the type 1 17beta-HSD gene. However, CTBs lying beneath the syncytial layer, e.g. those from which syncytiotrophoblasts develop, contained barely detectable amounts of type 1 17beta-HSD mRNA as determined by in situ hybridization. These findings, along with those from other laboratories confirm the primordial role of the syncytium in the synthesis of steroids during pregnancy. In addition, our results indicate for the first time that CTBs differentiating along the invasive pathway contain type 1 17beta-HSD mRNA.

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