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A Non-specific Setaria italica Lipid Transfer Protein Gene Plays a Critical Role under Abiotic Stress

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ABSTRACT

Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene (SiLTP) was isolated from a foxtail millet (Setaria italica) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis. Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP-OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo, respectively. Moreover, the SiLTP expression levels were higher in SiARDP-OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought tolerance levels.

No MeSH data available.


SiARDP regulates SiLTP expression by directly binding to the DRE element. EMSA for SiARDP and SiAREB1 binding to the promoter of SiLTP (A); yeast one-hybrid assay for SiARDP binding to the DRE element of the SiLTP promoter (B) and the SiLTP expression levels in SiARDP transgenic foxtail millet (C). Total RNA was extracted from 30 plants at the indicated times after the treatments. Data represent means and standard deviations for three biological replicates.
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Figure 10: SiARDP regulates SiLTP expression by directly binding to the DRE element. EMSA for SiARDP and SiAREB1 binding to the promoter of SiLTP (A); yeast one-hybrid assay for SiARDP binding to the DRE element of the SiLTP promoter (B) and the SiLTP expression levels in SiARDP transgenic foxtail millet (C). Total RNA was extracted from 30 plants at the indicated times after the treatments. Data represent means and standard deviations for three biological replicates.

Mentions: PlantCARE was used to analyze the promoter region of SiLTP, three cis-elements including two DRE and one AREB, were discovered (Supplementary Table S2) (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). EMSA was carried out and the result indicated that SiARDP bound to the SiLTP promoter through the DRE elements in vitro, but SiABRE1 could not bind to the AREB element (Figure 10A). Meanwhile, yeast one-hybrid results showed that SiARDP could bind to the SiLTP DRE elements in vivo (Figure 10B). The SiLTP transcription levels in SiARDP-OE lines were measured as well. The qPCR results showed that SiLTP was upregulated in SiARDP-OE lines (Li et al., 2014) by ∼1.5- to 2.0-fold compared with the WT (Figure 10C). Thus, SiLTP may be a downstream gene of SiARDP TF.


A Non-specific Setaria italica Lipid Transfer Protein Gene Plays a Critical Role under Abiotic Stress
SiARDP regulates SiLTP expression by directly binding to the DRE element. EMSA for SiARDP and SiAREB1 binding to the promoter of SiLTP (A); yeast one-hybrid assay for SiARDP binding to the DRE element of the SiLTP promoter (B) and the SiLTP expression levels in SiARDP transgenic foxtail millet (C). Total RNA was extracted from 30 plants at the indicated times after the treatments. Data represent means and standard deviations for three biological replicates.
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Figure 10: SiARDP regulates SiLTP expression by directly binding to the DRE element. EMSA for SiARDP and SiAREB1 binding to the promoter of SiLTP (A); yeast one-hybrid assay for SiARDP binding to the DRE element of the SiLTP promoter (B) and the SiLTP expression levels in SiARDP transgenic foxtail millet (C). Total RNA was extracted from 30 plants at the indicated times after the treatments. Data represent means and standard deviations for three biological replicates.
Mentions: PlantCARE was used to analyze the promoter region of SiLTP, three cis-elements including two DRE and one AREB, were discovered (Supplementary Table S2) (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). EMSA was carried out and the result indicated that SiARDP bound to the SiLTP promoter through the DRE elements in vitro, but SiABRE1 could not bind to the AREB element (Figure 10A). Meanwhile, yeast one-hybrid results showed that SiARDP could bind to the SiLTP DRE elements in vivo (Figure 10B). The SiLTP transcription levels in SiARDP-OE lines were measured as well. The qPCR results showed that SiLTP was upregulated in SiARDP-OE lines (Li et al., 2014) by ∼1.5- to 2.0-fold compared with the WT (Figure 10C). Thus, SiLTP may be a downstream gene of SiARDP TF.

View Article: PubMed Central - PubMed

ABSTRACT

Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene (SiLTP) was isolated from a foxtail millet (Setaria italica) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis. Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP-OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo, respectively. Moreover, the SiLTP expression levels were higher in SiARDP-OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought tolerance levels.

No MeSH data available.