Salicylic acid interferes with GFP fluorescence in vivo

Jonge, J. de; Hofius, Daniel; Hennig, Lars

doi:10.1093/jxb/erx031

Cited by 4 publications

(4 citation statements)

References 43 publications

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“…In addition, full-length CAMTA3 protein was detected in nuclei isolated from cold-treated plants and was at greater levels in these plants than it was in plants grown at warm temperature ( Figure 4E; a second band, marked with asterisk, was detected in the nuclear preparations, but not in the total protein preparations, indicating that it resulted from proteolytic cleavage of CAMTA3 during isolation of the nuclei). The small apparent discrepancy between the relative amounts of CAMTA3 protein in the nuclei of warm-and cold-treated plants observed in the fluorescence and immunoblotting assays was likely due to the interfering effects that SA has been reported to have on the fluorescence of GFP protein fusions in vivo (de Jonge et al, 2017). Taken together, our results indicated that the induction of SA pathway genes in cold-treated plants involved changes in the ability of the CAMTA3 protein to repress gene expression.…”

Section: Induction Of Sa Pathway Genes In Response To Low Temperaturementioning

confidence: 55%

CAMTA-Mediated Regulation of Salicylic Acid Immunity Pathway Genes in Arabidopsis Exposed to Low Temperature and Pathogen Infection

et al. 2017

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Arabidopsis thaliana calmodulin binding transcription activator (CAMTA) factors repress the expression of genes involved in salicylic acid (SA) biosynthesis and SA-mediated immunity in healthy plants grown at warm temperature (22°C). This repression is overcome in plants exposed to low temperature (4°C) for more than a week and in plants infected by biotrophic and hemibiotrophic pathogens. Here, we present evidence that CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves the action of an N-terminal repression module (NRM) that acts independently of calmodulin (CaM) binding to the IQ and CaM binding (CaMB) domains, a finding that is contrary to current thinking that CAMTA3 repression activity requires binding of CaM to the CaMB domain. Induction of SA pathway genes in response to low temperature did not occur in plants expressing only the CAMTA3-NRM region of the protein. Mutational analysis provided evidence that the repression activity of the NRM was suppressed by action of the IQ and CaMB domains responding to signals generated in response to low temperature. Plants expressing the CAMTA3-NRM region were also impaired in defense against the bacterial hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000. Our results indicate that the regulation of CAMTA3 repression activity by low temperature and pathogen infection involves related mechanisms, but with distinct differences.

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Section: Induction Of Sa Pathway Genes In Response To Low Temperaturementioning

confidence: 55%

CAMTA-Mediated Regulation of Salicylic Acid Immunity Pathway Genes in Arabidopsis Exposed to Low Temperature and Pathogen Infection

et al. 2017

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“…A DR5 signal depletion was detected in Arabidopsis roots upon 100-200 mM SA (Wang et al, 2007), but Zhao et al (2015) also reported a decrease in DR5 expression in the root tip upon 30 mM SA treatment. Even though 50-200 mM SA treatment inhibits GFP fluorescence (de Jonge et al, 2017), we consistently observe an increase in DR5::GFP fluorescence after 30 mM SA treatment. The results of de Jonge et al (2017) showed that the GFP in protein fusions, but not purified GFP, is affected by SA.…”

Section: Sa Modifies Auxin Distribution In the Root Tipmentioning

confidence: 60%

“…Even though 50-200 mM SA treatment inhibits GFP fluorescence (de Jonge et al, 2017), we consistently observe an increase in DR5::GFP fluorescence after 30 mM SA treatment. The results of de Jonge et al (2017) showed that the GFP in protein fusions, but not purified GFP, is affected by SA. However, the fact that DR5::GFP codes for free GFP does not explain this inconsistency, as we observe that TAA1:TAA1-GFP, a protein fusion, also shows an increase after SA treatment.…”

Section: Sa Modifies Auxin Distribution In the Root Tipmentioning

confidence: 60%

Salicylic Acid Affects Root Meristem Patterning via Auxin Distribution in a Concentration-Dependent Manner

et al. 2019

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The phytohormone salicylic acid (SA) is well known for its induction of pathogenesis-related proteins and systemic acquired resistance; SA also has specific effects on plant growth and development. Here we analyzed the effect of SA on Arabidopsis (Arabidopsis thaliana) root development. We show that exogenous SA treatment at low (below 50 mM) and high (greater than 50 mM) concentrations affect root meristem development in two different PR1-independent ways. Low-concentration SA promoted adventitious roots and altered architecture of the root apical meristem, whereas high-concentration SA inhibited all growth processes in the root. All exposures to exogenous SA led to changes in auxin synthesis and transport. A wide range of SA treatment concentrations activated auxin synthesis, but the effect of SA on auxin transport was dose dependent. Mathematical modeling of auxin synthesis and transport predicted auxin accumulation or depletion in the root tip following low-or highconcentration SA treatments, respectively. SA-induced auxin accumulation led to the formation of more layers of columella initials, an additional cortical cell layer (middle cortex), and extra files of epidermis, cortex, and endodermis cells. Suppression of SHORT ROOT and activation of CYCLIN D6;1 mediated the changes in radial architecture of the root. We propose that lowconcentration SA plays an important role in shaping root meristem structure and root system architecture.

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“…Factors causing this may include the amount of protein, 3 the pH associated with a proteinspecific localization 4,5 or interactions with other cellular components that destabilizes the fluorophore. 6 In addition, adding an endogenous protein tag of 25 to 80 KDa to a protein may generate dominant interactive effects that can interfere with protein activity and potentially affect the interpretation of the observed localization. 7,8 Here we use the recent localization of DEK1 (Defective kernel 1) in the moss Physcomitrella patens 9 to illustrate some of the pitfalls described above.…”

mentioning

confidence: 99%

Challenges of in vivo protein localization in plants seen through the DEK1 protein lens

Perroud

Demko

2020

Plant Signaling & Behavior

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During the last 25 y, fluorescent protein tagging has become a tool of choice to investigate protein function in a cellular context. The information gathered with this approach is not only providing insights into protein subcellular localization but also allows contextualizing protein function in multicellular settings. Here we illustrate the power of this method by commenting on the recent successful localization of the large membrane DEK1 protein during three-dimensional body formation in the moss Physcomitrella patens. But as many approaches, protein tagging is not exempt of caveats. The multiple infructuous (failed) attempts to detect DEK1 using a fluorescent protein tag present a good overview of such potential problems. Here we discuss the insertion of different fluorescent proteins at different positions in the PpDEK1 protein and the resulting unintended range of mutant phenotypes. Albeit none of these mutants generated a detectable fluorescent signal they can still provide interesting biological information about DEK1 function.

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Salicylic acid interferes with GFP fluorescence in vivo

Abstract: HighlightSalicylic acid (SA) interferes with fluorescence from GFP‐derived reporter constructs in planta and as such assays using these constructs with SA treatment should be evaluated cautiously.

Cited by 4 publications

References 43 publications

CAMTA-Mediated Regulation of Salicylic Acid Immunity Pathway Genes in Arabidopsis Exposed to Low Temperature and Pathogen Infection

CAMTA-Mediated Regulation of Salicylic Acid Immunity Pathway Genes in Arabidopsis Exposed to Low Temperature and Pathogen Infection

Salicylic Acid Affects Root Meristem Patterning via Auxin Distribution in a Concentration-Dependent Manner

Challenges of in vivo protein localization in plants seen through the DEK1 protein lens

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