scholarly journals Reconstitution of Cytokinin Signaling in Rice Protoplasts

2021 ◽  
Vol 22 (7) ◽  
pp. 3647
Author(s):  
Eunji Ga ◽  
Jaeeun Song ◽  
Myung Ki Min ◽  
Jihee Ha ◽  
Sangkyu Park ◽  
...  
Keyword(s):  
Expression System ◽  
Transient Gene Expression ◽  
Firefly Luciferase ◽  
Signaling Proteins ◽  
Type B ◽  
Response Regulators ◽  
Reporter Construct ◽  
Cytokinin Signaling ◽  
Firefly Luciferase Gene ◽  
Signaling Components

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

scholarly journals DEG9, a serine protease, modulates cytokinin and light signaling by regulating the level of ARABIDOPSIS RESPONSE REGULATOR 4

2016 ◽  
Vol 113 (25) ◽  
pp. E3568-E3576 ◽  
Author(s):  
Wei Chi ◽  
Jing Li ◽  
Baoye He ◽  
Xin Chai ◽  
Xiumei Xu ◽  
...  
Keyword(s):  
Serine Protease ◽  
Response Regulator ◽  
Light Signaling ◽  
Plant Signaling ◽  
Response Regulators ◽  
Cytokinin Signaling ◽  
Periplasmic Proteins ◽  
The Stability ◽  
Protease Degradation ◽  
Signaling Components

Cytokinin is an essential phytohormone that controls various biological processes in plants. A number of response regulators are known to be important for cytokinin signal transduction. ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4) mediates the cross-talk between light and cytokinin signaling through modulation of the activity of phytochrome B. However, the mechanism that regulates the activity and stability of ARR4 is unknown. Here we identify an ATP-independent serine protease, degradation of periplasmic proteins 9 (DEG9), which localizes to the nucleus and regulates the stability of ARR4. Biochemical evidence shows that DEG9 interacts with ARR4, thereby targeting ARR4 for degradation, which suggests that DEG9 regulates the stability of ARR4. Moreover, genetic evidence shows that DEG9 acts upstream of ARR4 and regulates the activity of ARR4 in cytokinin and light-signaling pathways. This study thus identifies a role for a ubiquitin-independent selective protein proteolysis in the regulation of the stability of plant signaling components.

scholarly journals SCFKMD controls cytokinin signaling by regulating the degradation of type-B response regulators

2013 ◽  
Vol 110 (24) ◽  
pp. 10028-10033 ◽  
Author(s):  
H. J. Kim ◽  
Y.-H. Chiang ◽  
J. J. Kieber ◽  
G. E. Schaller
Keyword(s):  
Type B ◽  
Response Regulators ◽  
Cytokinin Signaling

scholarly journals Type-B response regulators of rice play key roles in growth, development and cytokinin signaling

Development ◽  
2019 ◽  
Vol 146 (13) ◽  
pp. dev174870 ◽  
Author(s):  
Jennifer M. Worthen ◽  
Maria V. Yamburenko ◽  
Jeewoo Lim ◽  
Zachary L. Nimchuk ◽  
Joseph J. Kieber ◽  
...  
Keyword(s):  
Type B ◽  
Response Regulators ◽  
Cytokinin Signaling ◽  
Growth Development

scholarly journals Type B Response Regulators of Arabidopsis Play Key Roles in Cytokinin Signaling and Plant Development

2008 ◽  
Vol 20 (8) ◽  
pp. 2102-2116 ◽  
Author(s):  
Rebecca D. Argyros ◽  
Dennis E. Mathews ◽  
Yi-Hsuan Chiang ◽  
Christine M. Palmer ◽  
Derek M. Thibault ◽  
...  
Keyword(s):  
Plant Development ◽  
Type B ◽  
Response Regulators ◽  
Cytokinin Signaling

Cytokinin Signaling Is Essential for Organ Formation in Marchantia polymorpha

2019 ◽  
Vol 60 (8) ◽  
pp. 1842-1854 ◽  
Author(s):  
Shiori S Aki ◽  
Tatsuya Mikami ◽  
Satoshi Naramoto ◽  
Ryuichi Nishihama ◽  
Kimitsune Ishizaki ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Marchantia Polymorpha ◽  
Cytokinin Oxidase ◽  
Endogenous Cytokinin ◽  
Type B ◽  
Response Regulators ◽  
Cytokinin Signaling ◽  
Organ Formation ◽  
Cytokinin Treatment ◽  
Rhizoid Formation

Abstract Cytokinins are known to regulate various physiological events in plants. Cytokinin signaling is mediated by the phosphorelay system, one of the most ancient mechanisms controlling hormonal pathways in plants. The liverwort Marchantia polymorpha possesses all components necessary for cytokinin signaling; however, whether they respond to cytokinins and how the signaling is fine-tuned remain largely unknown. Here, we report cytokinin function in Marchantia development and organ formation. Our measurement of cytokinin species revealed that cis-zeatin is the most abundant cytokinin in Marchantia. We reduced the endogenous cytokinin level by overexpressing the gene for cytokinin oxidase, MpCKX, which inactivates cytokinins, and generated overexpression and knockout lines for type-A (MpRRA) and type-B (MpRRB) response regulators to manipulate the signaling. The overexpression lines of MpCKX and MpRRA, and the knockout lines of MpRRB, shared phenotypes such as inhibition of gemma cup formation, enhanced rhizoid formation and hyponastic thallus growth. Conversely, the knockout lines of MpRRA produced more gemma cups and exhibited epinastic thallus growth. MpRRA expression was elevated by cytokinin treatment and reduced by knocking out MpRRB, suggesting that MpRRA is upregulated by the MpRRB-mediated cytokinin signaling, which is antagonized by MpRRA. Our findings indicate that when plants moved onto land they already deployed the negative feedback loop of cytokinin signaling, which has an indispensable role in organogenesis.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1661-1671
Author(s):  
Klaus Maleck ◽  
Urs Neuenschwander ◽  
Rebecca M Cade ◽  
Robert A Dietrich ◽  
Jeffery L Dangl ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Acquired Resistance ◽  
Constitutive Expression ◽  
Firefly Luciferase ◽  
Marker Genes ◽  
Arabidopsis Mutants ◽  
Isolation And Characterization ◽  
Firefly Luciferase Gene

Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

scholarly journals The Relationship between AGAMOUS and Cytokinin Signaling in the Establishment of Carpeloid Features

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 827
Author(s):  
Andrea Gómez-Felipe ◽  
Daniel Kierzkowski ◽  
Stefan de Folter
Keyword(s):  
Regulatory Network ◽  
Dependent Manner ◽  
Mads Box ◽  
Type B ◽  
Cytokinin Signaling ◽  
Reproductive Structures ◽  
Carpel Development ◽  
Gene Regulatory ◽  
The Relationship ◽  
Gynoecium Development

Gynoecium development is dependent on gene regulation and hormonal pathway interactions. The phytohormones auxin and cytokinin are involved in many developmental programs, where cytokinin is normally important for cell division and meristem activity, while auxin induces cell differentiation and organ initiation in the shoot. The MADS-box transcription factor AGAMOUS (AG) is important for the development of the reproductive structures of the flower. Here, we focus on the relationship between AG and cytokinin in Arabidopsis thaliana, and use the weak ag-12 and the strong ag-1 allele. We found that cytokinin induces carpeloid features in an AG-dependent manner and the expression of the transcription factors CRC, SHP2, and SPT that are involved in carpel development. AG is important for gynoecium development, and contributes to regulating, or else directly regulates CRC, SHP2, and SPT. All four genes respond to either reduced or induced cytokinin signaling and have the potential to be regulated by cytokinin via the type-B ARR proteins. We generated a model of a gene regulatory network, where cytokinin signaling is mainly upstream and in parallel with AG activity.

scholarly journals Assembly of Two Transgenes in an Artificial Chromatin Domain Gives Highly Coordinated Expression in Tobacco

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 727-740
Author(s):  
Ludmila Mlynárová ◽  
Annelies Loonen ◽  
Elzbieta Mietkiewska ◽  
Ritsert C Jansen ◽  
Jan-Peter Nap
Keyword(s):  
Enzyme Activities ◽  
Firefly Luciferase ◽  
Chromatin Domain ◽  
Mrna Levels ◽  
Loop Model ◽  
Data Generation ◽  
Genome Data ◽  
The Matrix ◽  
Coordinated Expression ◽  
Firefly Luciferase Gene

Abstract The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli β-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tabacum). In plants carrying A elements, quantitative enzyme activities and mRNA levels of both genes show high correlations compared to control plants. The A element thus creates an artificial chromatin domain that yields coordinated expression. Surprisingly, enzyme activities correlated poorly with their respective mRNA levels. We hypothesize that this indicates the occurrence of “error pipelines” in data generation: systematic errors of a given analytical method will point in the same direction and cancel out in correlation analysis, resulting in better correlations. In combining different methods of analysis, however, such errors do not cancel out and as a result relevant correlations can be masked. Such error pipelines will have to be taken into account when different types of (e.g., whole-genome) data sets are combined in quantitative analyses.

Sign in / Sign up

Export Citation Format

Share Document