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Characterization and co-expression analysis of WRKY orthologs involved in responses to multiple abiotic stresses in Pak-choi (Brassica campestris ssp. chinensis).

Tang J, Wang F, Wang Z, Huang Z, Xiong A, Hou X - BMC Plant Biol. (2013)

Bottom Line: Experiments involving BcWRKY25 and BcWRKY40 confirmed the prediction.A total of 22 stress-inducible BcWRKYs found in leaves can co-regulate multiple environmental stresses by integrating the potential mutual interactions of WRKYs in Pak-choi.This information will be valuable when exploring the molecular mechanisms of WRKYs in response to abiotic stresses in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China. hxl@njau.edu.cn.

ABSTRACT

Background: The WRKY transcription factor is an important member of the stress-related transcription factors, which mediate diverse abiotic stresses in many plants. However, up until now, the number of WRKY members, and the regulatory mechanisms involved in abiotic stress responses in Pak-choi (Brassica campestris ssp. chinensis), remained unknown.

Results: We isolated and identified 56 full-length WRKY cDNAs from a Pak-choi stress-induced cDNA library. The 56 putative BcWRKY proteins were divided into three groups based on structural and phylogenetic analyses. A subcellular localization prediction indicated that the putative BcWRKY proteins were enriched in the nuclear region. Experiments involving BcWRKY25 and BcWRKY40 confirmed the prediction. A total of 22 BcWRKYs were differentially expressed in response to at least one stress condition (abscisic acid, cold, salinity, heat, or osmosis) tested on Pak-choi leaves, and a co-expression analysis indicated stress-inducible BcWRKYs co-regulated multiple abiotic stresses. BcWRKY33, BcWRKY40, BcWRKY53, and BcWRKY70 acted as key regulators and played dominant roles within co-regulatory networks of stress-inducible BcWRKYs.

Conclusions: We first isolated and characterized the 56 stress-inducible WRKY transcription factor family members. A total of 22 stress-inducible BcWRKYs found in leaves can co-regulate multiple environmental stresses by integrating the potential mutual interactions of WRKYs in Pak-choi. This information will be valuable when exploring the molecular mechanisms of WRKYs in response to abiotic stresses in plants.

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Subcellular localization analysis of BcWRKYs. The subcellular localization analysis of the putative BcWRKY proteins used NLStradamus, NucPred, and WOLF PSORT with the default settings to detect nuclear localization scores. (A) two yellow fluorescent protein (YFP) marker expressing BcWRKY fusion genes, BcWRKY25-YFP and BcWRKY40-YFP, were constructed; (B) BcWRKY25-YFP and BcWRKY40-YFP were introduced into onion epidermal cells by particle bombardment with the YFP signal as an indicating marker to test the subcellular localization of BcWRKY proteins; (C) the upper panel, the corresponding bright field, fluorescence, merged fluorescence image of YFP control; the middle panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW25-YFP; the lower panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW40-YFP. Scale bars: 20 μm.
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Figure 3: Subcellular localization analysis of BcWRKYs. The subcellular localization analysis of the putative BcWRKY proteins used NLStradamus, NucPred, and WOLF PSORT with the default settings to detect nuclear localization scores. (A) two yellow fluorescent protein (YFP) marker expressing BcWRKY fusion genes, BcWRKY25-YFP and BcWRKY40-YFP, were constructed; (B) BcWRKY25-YFP and BcWRKY40-YFP were introduced into onion epidermal cells by particle bombardment with the YFP signal as an indicating marker to test the subcellular localization of BcWRKY proteins; (C) the upper panel, the corresponding bright field, fluorescence, merged fluorescence image of YFP control; the middle panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW25-YFP; the lower panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW40-YFP. Scale bars: 20 μm.

Mentions: To investigate the subcellular localization of putative BcWRKY proteins, we used NLStradamus with the default settings. We found 50 BcWRKY proteins contained NLSs. Additionally, we used NucPred and WOLF PSORT to predict the nuclear localization scores of the BcWRKY proteins. Fifty BcWRKY proteins had a NucPres-score of ≥ 0.5 and 53 had nuclear localization scores of ≥ 7 (KNN = 14) using WOLF PSORT (Table 1). A consensus of the results generated predicted that most BcWRKYs (47/56) localized at the nucleus (Figure 3A). Additionally, we used a transient expression system in onion epidermal cells to test the subcellular localization of BcWRKY proteins. The yellow fluorescent marker protein (YFP) was fused to BcWRKY25 and BcWRKY40 and the expression of the fusion genes was tracked by the marker’s signal (Figure 3B). When YFP alone was expressed the fluorescence was observed in the cytosol and nucleus (Figure 3C, upper panel), while the yellow fluorescence of the BcWRKY25-YFP and BcWRKY40-YFP fusions were observed in the nuclear region (Figure 3C, middle and lower panel, respectively). Thus, BcWRKY25 and 40 were localized to the nucleus, which agreed with the protein subcellular localization prediction. These results indicate that the properties of the BcWRKY proteins define them as transcription factors.


Characterization and co-expression analysis of WRKY orthologs involved in responses to multiple abiotic stresses in Pak-choi (Brassica campestris ssp. chinensis).

Tang J, Wang F, Wang Z, Huang Z, Xiong A, Hou X - BMC Plant Biol. (2013)

Subcellular localization analysis of BcWRKYs. The subcellular localization analysis of the putative BcWRKY proteins used NLStradamus, NucPred, and WOLF PSORT with the default settings to detect nuclear localization scores. (A) two yellow fluorescent protein (YFP) marker expressing BcWRKY fusion genes, BcWRKY25-YFP and BcWRKY40-YFP, were constructed; (B) BcWRKY25-YFP and BcWRKY40-YFP were introduced into onion epidermal cells by particle bombardment with the YFP signal as an indicating marker to test the subcellular localization of BcWRKY proteins; (C) the upper panel, the corresponding bright field, fluorescence, merged fluorescence image of YFP control; the middle panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW25-YFP; the lower panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW40-YFP. Scale bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222839&req=5

Figure 3: Subcellular localization analysis of BcWRKYs. The subcellular localization analysis of the putative BcWRKY proteins used NLStradamus, NucPred, and WOLF PSORT with the default settings to detect nuclear localization scores. (A) two yellow fluorescent protein (YFP) marker expressing BcWRKY fusion genes, BcWRKY25-YFP and BcWRKY40-YFP, were constructed; (B) BcWRKY25-YFP and BcWRKY40-YFP were introduced into onion epidermal cells by particle bombardment with the YFP signal as an indicating marker to test the subcellular localization of BcWRKY proteins; (C) the upper panel, the corresponding bright field, fluorescence, merged fluorescence image of YFP control; the middle panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW25-YFP; the lower panel, the corresponding bright field, fluorescence, merged fluorescence image of BcW40-YFP. Scale bars: 20 μm.
Mentions: To investigate the subcellular localization of putative BcWRKY proteins, we used NLStradamus with the default settings. We found 50 BcWRKY proteins contained NLSs. Additionally, we used NucPred and WOLF PSORT to predict the nuclear localization scores of the BcWRKY proteins. Fifty BcWRKY proteins had a NucPres-score of ≥ 0.5 and 53 had nuclear localization scores of ≥ 7 (KNN = 14) using WOLF PSORT (Table 1). A consensus of the results generated predicted that most BcWRKYs (47/56) localized at the nucleus (Figure 3A). Additionally, we used a transient expression system in onion epidermal cells to test the subcellular localization of BcWRKY proteins. The yellow fluorescent marker protein (YFP) was fused to BcWRKY25 and BcWRKY40 and the expression of the fusion genes was tracked by the marker’s signal (Figure 3B). When YFP alone was expressed the fluorescence was observed in the cytosol and nucleus (Figure 3C, upper panel), while the yellow fluorescence of the BcWRKY25-YFP and BcWRKY40-YFP fusions were observed in the nuclear region (Figure 3C, middle and lower panel, respectively). Thus, BcWRKY25 and 40 were localized to the nucleus, which agreed with the protein subcellular localization prediction. These results indicate that the properties of the BcWRKY proteins define them as transcription factors.

Bottom Line: Experiments involving BcWRKY25 and BcWRKY40 confirmed the prediction.A total of 22 stress-inducible BcWRKYs found in leaves can co-regulate multiple environmental stresses by integrating the potential mutual interactions of WRKYs in Pak-choi.This information will be valuable when exploring the molecular mechanisms of WRKYs in response to abiotic stresses in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China. hxl@njau.edu.cn.

ABSTRACT

Background: The WRKY transcription factor is an important member of the stress-related transcription factors, which mediate diverse abiotic stresses in many plants. However, up until now, the number of WRKY members, and the regulatory mechanisms involved in abiotic stress responses in Pak-choi (Brassica campestris ssp. chinensis), remained unknown.

Results: We isolated and identified 56 full-length WRKY cDNAs from a Pak-choi stress-induced cDNA library. The 56 putative BcWRKY proteins were divided into three groups based on structural and phylogenetic analyses. A subcellular localization prediction indicated that the putative BcWRKY proteins were enriched in the nuclear region. Experiments involving BcWRKY25 and BcWRKY40 confirmed the prediction. A total of 22 BcWRKYs were differentially expressed in response to at least one stress condition (abscisic acid, cold, salinity, heat, or osmosis) tested on Pak-choi leaves, and a co-expression analysis indicated stress-inducible BcWRKYs co-regulated multiple abiotic stresses. BcWRKY33, BcWRKY40, BcWRKY53, and BcWRKY70 acted as key regulators and played dominant roles within co-regulatory networks of stress-inducible BcWRKYs.

Conclusions: We first isolated and characterized the 56 stress-inducible WRKY transcription factor family members. A total of 22 stress-inducible BcWRKYs found in leaves can co-regulate multiple environmental stresses by integrating the potential mutual interactions of WRKYs in Pak-choi. This information will be valuable when exploring the molecular mechanisms of WRKYs in response to abiotic stresses in plants.

Show MeSH
Related in: MedlinePlus