scholarly journals Limitation of Cytokinin Export to the Shoots by Nucleoside Transporter ENT3 and Its Linkage with Root Elongation in Arabidopsis

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 350
Author(s):  
Alla Korobova ◽  
Bulat Kuluev ◽  
Torsten Möhlmann ◽  
Dmitriy Veselov ◽  
Guzel Kudoyarova
Keyword(s):  
Root Elongation ◽  
Zeatin Riboside ◽  
Mineral Nutrient ◽  
Endogenous Cytokinin ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Root Cells ◽  
Dna Insertion ◽  
Endogenous Cytokinins

The trans-membrane carrier AtENT3 is known to transport externally supplied cytokinin ribosides and thus promote uptake by cells. However, its role in distributing either exogenous or endogenous cytokinins within the intact plant has not hitherto been reported. To test this, we used ent3-1 mutant Arabidopsis seedlings in which the gene is not expressed due to a T-DNA insertion, and examined the effect on the concentration and distribution of either endogenous cytokinins or exogenous trans-zeatin riboside applied to the roots. In the mutant, accumulation of endogenous cytokinins in the roots was reduced and capacity to deliver externally supplied trans-zeatin riboside to the shoots was increased suggesting involvement of equilibrative nucleoside (ENT) transporter in the control of cytokinin distribution in the plants. Roots of ent3-1 were longer in the mutant in association with their lower cytokinin concentration. We concluded that the ENT3 transporter participates in partitioning endogenous cytokinins between the apoplast and the symplast by facilitating their uptake by root cells thereby limiting cytokinin export to the shoots through the xylem. Dilution of the mineral nutrient solution lowered endogenous cytokinin concentration in the roots of both wild type (WT) and ent3-1 plants accompanied by promotion of root elongation. Nevertheless, cytokinin content was lower, while roots were longer in the ent3-1 mutant than in the WT under either normal or deficient mineral nutrition suggesting a significant role of ENT3 transporter in the control of cytokinin level in the roots and the rate of their elongation.

scholarly journals PARTICIPATION OF ENT3 TRANSPORTER IN CYTOKININ DISTRIBUTION BETWEEN THE ROOT AND THE SHOOT AND IN REGULATION OF GROWTH RESPONSE OF ARABIDOPSIS PLANTS TO INCREASED LEVEL OF MINERAL NUTRITION

ÈKOBIOTEH ◽  
2020 ◽  
Vol 3 (4) ◽  
pp. 741-749
Author(s):  
A.V. Korobova ◽  
◽  
Z.A. Akhtyamova ◽  
B.R. Kuluev ◽  
G.R. Kudoyarova ◽  
...  
Keyword(s):  
Mineral Nutrition ◽  
Root Elongation ◽  
Growth Response ◽  
Zeatin Riboside ◽  
Root Cells ◽  
Endogenous Cytokinins ◽  
Nitrogenous Base ◽  
Regulation Of Growth ◽  
Adaptive Role ◽  
Inhibition Of Root Elongation

The nitrogenous base riboside transporter ENT3 is known for its ability to transport ribosylated cytokinins across the membrane. However, its role in the distribution of cytokinins between plant organs has not yet been studied. For this purpose, we compared the content and distribution of either endogenous cytokinins or the exogenous trans-zeatin riboside introduced into the nutrient solution in mutant ent3-1 plants and the parent genotype Columbia. In the mutant, the accumulation of endogenous cytokinins in the roots was suppressed, and the ability to deliver exogenous trans-zeatin riboside to shoots increased. The roots of ent3-1 were about 15% longer and had a lower cytokinin concentration. A thirtyfold increase in the concentration of macronutrients led to inhibition of root elongation in the original Columbia line, but not in ent3-1 plants. This growth response occured in accordance with the content of cytokinins in the roots: in Columbia plants, cytokinins accumulated in these organs. The increase in the level of hormones in the roots of ent3-1 was to a lesser extent and was not significant. It was concluded that the ENT3 transporter is involved in the distribution of endogenous cytokinins between the apoplast and symplast, facilitating their uptake by root cells, thereby limiting the export of cytokinins to shoots through the xylem, and can play an important adaptive role in changing the level of mineral nutrition.

scholarly journals Understanding the Role of Cytokinins in Tissue Proliferation of Rhododendron `Montego'

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 447B-447
Author(s):  
Eric W. Mercure ◽  
Carol A. Auer ◽  
Mark H. Brand
Keyword(s):  
Multiple Shoots ◽  
Shoot Formation ◽  
Zeatin Riboside ◽  
Shoot Cultures ◽  
Tissue Proliferation ◽  
Exogenous Cytokinin ◽  
Endogenous Cytokinins ◽  
Extensive Degradation

Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons propagated in vitro. However, TP of Rhododendron `Montego' is observed initially in in vitro shoot cultures and it is characterized by the formation of multiple shoots with small leaves and nodal tumors. The formation of shoots in `Montego' TP (TP+) shoot cultures occurs without the presence of exogenous cytokinin in the medium, unlike normal `Montego' (TP–) shoot cultures, which require cytokinin for shoot growth. Structural studies have shown that tumors are composed of many adventitious buds and parenchyma cells, suggesting that TP is a result of abnormal cytokinin regulation that is controlling tumor and shoot formation. Two approaches are being used to determine if differences in cytokinin concentration and/or metabolism exist between TP+ and TP– shoot cultures. In the first approach, shoot cultures are grown in vitro for 1 week in the presence of tritiated isopentenyladenine (iP). Cytokinin uptake and metabolism are analyzed using HPLC and other analytical methods. Experiments suggest that extensive degradation and N-glucoside conjugation occur in TP+ and TP– shoots, resulting in the removal of most of the exogenous iP. In the second approach, the levels of endogenous cytokinins such as iP, isopentenyladenosine, zeatin, and zeatin riboside, are being measured in TP+ tumors and shoots and in TP– shoots by an ELISA method.

scholarly journals Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Qi Jia ◽  
Paul Bundock ◽  
Paul J. J. Hooykaas ◽  
Sylvia de Pater
Keyword(s):  
Gene Targeting ◽  
Wild Type ◽  
End Joining ◽  
Dsb Repair ◽  
Critical Pathway ◽  
Dna Integration ◽  
Dna Insertion ◽  
Non Homologous End Joining ◽  
The Stability

In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

scholarly journals Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection

2010 ◽  
Vol 298 (3) ◽  
pp. H771-H777 ◽  
Author(s):  
Jennifer B. Rose ◽  
Zlatina Naydenova ◽  
Andrew Bang ◽  
Megumi Eguchi ◽  
Gary Sweeney ◽  
...  
Keyword(s):  
Essential Role ◽  
Expression Profiles ◽  
Receptor Expression ◽  
Purine Nucleoside ◽  
Nucleoside Transport ◽  
Null Mouse ◽  
Mouse Heart ◽  
Nucleoside Transporter ◽  
Wild Type

To better understand the role of equilibrative nucleoside transporters (ENT) in purine nucleoside-dependent physiology of the cardiovascular system, we investigated whether the ENT1-null mouse heart was cardioprotected in response to ischemia (coronary occlusion for 30 min followed by reperfusion for 2 h). We observed that ENT1-null mouse hearts showed significantly less myocardial infarction compared with wild-type littermates. We confirmed that isolated wild-type adult mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit severely impaired nucleoside transport and lack ENT1 transcript and protein expression. Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4 were similar in cardiomyocytes isolated from ENT1-null adult mice compared with cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA knockdown of ENT1 in the cardiomyocyte cell line, HL-1, mimics findings in ENT1-null cardiomyocytes. Taken together, our data demonstrate that ENT1 plays an essential role in cardioprotection, most likely due to its effects in modulating purine nucleoside-dependent signaling and that the ENT1-null mouse is a powerful model system for the study of the role of ENTs in the physiology of the cardiomyocyte.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1)

2018 ◽  
Vol 475 (20) ◽  
pp. 3293-3309 ◽  
Author(s):  
Sylvia Y.M. Yao ◽  
Amy M.L. Ng ◽  
Carol E. Cass ◽  
James D. Young
Keyword(s):  
Expression System ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Binding Characteristics ◽  
Equilibrative Nucleoside Transporter ◽  
C Terminus ◽  
Nucleoside Drugs

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

scholarly journals Salicylic Acid Accumulation Controlled by LSD1 Is Essential in Triggering Cell Death in Response to Abiotic Stress

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 962
Author(s):  
Maciej Jerzy Bernacki ◽  
Anna Rusaczonek ◽  
Weronika Czarnocka ◽  
Stanisław Karpiński
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Abiotic Stress ◽  
Wild Type ◽  
Pr Genes ◽  
Genes Expression ◽  
Salicylic Acid Accumulation ◽  
Cell Death Phenotype ◽  
Insight Into

Salicylic acid (SA) is well known hormonal molecule involved in cell death regulation. In response to a broad range of environmental factors (e.g., high light, UV, pathogens attack), plants accumulate SA, which participates in cell death induction and spread in some foliar cells. LESION SIMULATING DISEASE 1 (LSD1) is one of the best-known cell death regulators in Arabidopsis thaliana. The lsd1 mutant, lacking functional LSD1 protein, accumulates SA and is conditionally susceptible to many biotic and abiotic stresses. In order to get more insight into the role of LSD1-dependent regulation of SA accumulation during cell death, we crossed the lsd1 with the sid2 mutant, caring mutation in ISOCHORISMATE SYNTHASE 1(ICS1) gene and having deregulated SA synthesis, and with plants expressing the bacterial nahG gene and thus decomposing SA to catechol. In response to UV A+B irradiation, the lsd1 mutant exhibited clear cell death phenotype, which was reversed in lsd1/sid2 and lsd1/NahG plants. The expression of PR-genes and the H2O2 content in UV-treated lsd1 were significantly higher when compared with the wild type. In contrast, lsd1/sid2 and lsd1/NahG plants demonstrated comparability with the wild-type level of PR-genes expression and H2O2. Our results demonstrate that SA accumulation is crucial for triggering cell death in lsd1, while the reduction of excessive SA accumulation may lead to a greater tolerance toward abiotic stress.

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