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A receptor-like kinase gene (GbRLK) from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis.

Zhao J, Gao Y, Zhang Z, Chen T, Guo W, Zhang T - BMC Plant Biol. (2013)

Bottom Line: Transgenic Arabidopsis with constitutive overexpression of GbRLK exhibited a reduced rate of water loss in leaves in vitro, along with improved salinity and drought tolerance and increased sensitivity to ABA compared with non-transgenic Col-0 Arabidopsis.Overexpression of GbRLK may improve stress tolerance by regulating stress-responsive genes to reduce water loss.Further studying of GbRLK will help elucidate abiotic stress signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Key Laboratory of Crop Genetics & Germplasm Enhancement, MOE Hybrid Cotton R&D Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China.

ABSTRACT

Background: Cotton (Gossypium spp.) is widely cultivated due to the important economic value of its fiber. However, extreme environmental degradation impedes cotton growth and production. Receptor-like kinase (RLK) proteins play important roles in signal transduction and participate in a diverse range of processes in response to plant hormones and environmental cues. Here, we introduced an RLK gene (GbRLK) from cotton into Arabidopsis and investigated its role in imparting abiotic stress tolerance.

Results: GbRLK transcription was induced by exogenously supplied abscisic acid (ABA), salicylic acid, methyl jasmonate, mock drought conditions and high salinity. We cloned the promoter sequence of this gene via self-formed adaptor PCR. Sequence analysis revealed that the promoter region contains many cis-acting stress-responsive elements such as ABRE, W-Box, MYB-core, W-Box core, TCA-element and others. We constructed a vector containing a 1,890-bp sequence in the 5' region upstream of the initiation codon of this promoter and transformed it into Arabidopsis thaliana. GUS histochemical staining analysis showed that GbRLK was expressed mainly in leaf veins, petioles and roots of transgenic Arabidopsis, but not in the cotyledons or root hairs. GbRLK promoter activity was induced by ABA, PEG, NaCl and Verticillium dahliae. Transgenic Arabidopsis with constitutive overexpression of GbRLK exhibited a reduced rate of water loss in leaves in vitro, along with improved salinity and drought tolerance and increased sensitivity to ABA compared with non-transgenic Col-0 Arabidopsis. Expression analysis of stress-responsive genes in GbRLK Arabidopsis revealed that there was increased expression of genes involved in the ABA-dependent signaling pathway (AtRD20, AtRD22 and AtRD26) and antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1) but not ion transporter genes (AtNHX1, AtSOS1).

Conclusions: GbRLK is involved in the drought and high salinity stresses pathway by activating or participating in the ABA signaling pathway. Overexpression of GbRLK may improve stress tolerance by regulating stress-responsive genes to reduce water loss. GbRLK may be employed in the genetic engineering of novel cotton cultivars in the future. Further studying of GbRLK will help elucidate abiotic stress signaling pathways.

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The expression of stress-responsive genes in transgenic GbRLK Arabidopsis vs. nontransgenic Col-0. Relative expression levels of stress-responsive genes were determined by qRT-PCR using cDNA synthesized from total RNAs isolated from the leaves of 2-week-old Arabidopsis grown in soil under normal conditions. Relative gene expression levels were determined using the 2^-ΔΔCT method. The CT (cycle threshold) values for both the target and internal control genes were means of three technical replicates. ΔCt = Ct target gene-CtRuBisCo. ΔΔCT = (CTtarget -CTRuBisCo)Transgenic - (CTtarget - CTRuBisCo) Non-transgenic. The large subunit of the RuBisCo gene (AtRuBisCo-F3/R3; Additional file1: Table S1) from Arabidopsis was used as an internal control for normalization of different cDNA samples. The primers for the amplification of stress-responsive genes (Additional file1: Table S1) were design based on sequences obtained from NCBI using Primer 5.0 software. Error bars represent standard error of means based on three independent reactions.
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Figure 6: The expression of stress-responsive genes in transgenic GbRLK Arabidopsis vs. nontransgenic Col-0. Relative expression levels of stress-responsive genes were determined by qRT-PCR using cDNA synthesized from total RNAs isolated from the leaves of 2-week-old Arabidopsis grown in soil under normal conditions. Relative gene expression levels were determined using the 2^-ΔΔCT method. The CT (cycle threshold) values for both the target and internal control genes were means of three technical replicates. ΔCt = Ct target gene-CtRuBisCo. ΔΔCT = (CTtarget -CTRuBisCo)Transgenic - (CTtarget - CTRuBisCo) Non-transgenic. The large subunit of the RuBisCo gene (AtRuBisCo-F3/R3; Additional file1: Table S1) from Arabidopsis was used as an internal control for normalization of different cDNA samples. The primers for the amplification of stress-responsive genes (Additional file1: Table S1) were design based on sequences obtained from NCBI using Primer 5.0 software. Error bars represent standard error of means based on three independent reactions.

Mentions: To reveal the possible molecular mechanisms underlying the improvement of stress resistance and tolerance in transgenic Arabidopsis thaliana by GbRLK, we studied the expression patterns of stress-responsive genes (AtRD20, AtRD22 and AtRD26), ion transporter genes (AtNHX1 and AtSOS1) and antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1) in transgenic and nontransgenic Arabidopsis thaliana. We examined the expression of these genes in three Arabidopsis lines, including two genetically pure clones, K-6 and K-2, which exhibited differences in stress resistance, and the nontransgenic line Col-0. The expression of all of the genes, except for ion transporter genes (AtNHX1, AtSOS1), was upregulated in transgenic GbRLK plants compared with Col-0 plants, especially antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1), which exhibited higher levels of upregulation than other genes (Figure 6).


A receptor-like kinase gene (GbRLK) from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis.

Zhao J, Gao Y, Zhang Z, Chen T, Guo W, Zhang T - BMC Plant Biol. (2013)

The expression of stress-responsive genes in transgenic GbRLK Arabidopsis vs. nontransgenic Col-0. Relative expression levels of stress-responsive genes were determined by qRT-PCR using cDNA synthesized from total RNAs isolated from the leaves of 2-week-old Arabidopsis grown in soil under normal conditions. Relative gene expression levels were determined using the 2^-ΔΔCT method. The CT (cycle threshold) values for both the target and internal control genes were means of three technical replicates. ΔCt = Ct target gene-CtRuBisCo. ΔΔCT = (CTtarget -CTRuBisCo)Transgenic - (CTtarget - CTRuBisCo) Non-transgenic. The large subunit of the RuBisCo gene (AtRuBisCo-F3/R3; Additional file1: Table S1) from Arabidopsis was used as an internal control for normalization of different cDNA samples. The primers for the amplification of stress-responsive genes (Additional file1: Table S1) were design based on sequences obtained from NCBI using Primer 5.0 software. Error bars represent standard error of means based on three independent reactions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750506&req=5

Figure 6: The expression of stress-responsive genes in transgenic GbRLK Arabidopsis vs. nontransgenic Col-0. Relative expression levels of stress-responsive genes were determined by qRT-PCR using cDNA synthesized from total RNAs isolated from the leaves of 2-week-old Arabidopsis grown in soil under normal conditions. Relative gene expression levels were determined using the 2^-ΔΔCT method. The CT (cycle threshold) values for both the target and internal control genes were means of three technical replicates. ΔCt = Ct target gene-CtRuBisCo. ΔΔCT = (CTtarget -CTRuBisCo)Transgenic - (CTtarget - CTRuBisCo) Non-transgenic. The large subunit of the RuBisCo gene (AtRuBisCo-F3/R3; Additional file1: Table S1) from Arabidopsis was used as an internal control for normalization of different cDNA samples. The primers for the amplification of stress-responsive genes (Additional file1: Table S1) were design based on sequences obtained from NCBI using Primer 5.0 software. Error bars represent standard error of means based on three independent reactions.
Mentions: To reveal the possible molecular mechanisms underlying the improvement of stress resistance and tolerance in transgenic Arabidopsis thaliana by GbRLK, we studied the expression patterns of stress-responsive genes (AtRD20, AtRD22 and AtRD26), ion transporter genes (AtNHX1 and AtSOS1) and antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1) in transgenic and nontransgenic Arabidopsis thaliana. We examined the expression of these genes in three Arabidopsis lines, including two genetically pure clones, K-6 and K-2, which exhibited differences in stress resistance, and the nontransgenic line Col-0. The expression of all of the genes, except for ion transporter genes (AtNHX1, AtSOS1), was upregulated in transgenic GbRLK plants compared with Col-0 plants, especially antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1), which exhibited higher levels of upregulation than other genes (Figure 6).

Bottom Line: Transgenic Arabidopsis with constitutive overexpression of GbRLK exhibited a reduced rate of water loss in leaves in vitro, along with improved salinity and drought tolerance and increased sensitivity to ABA compared with non-transgenic Col-0 Arabidopsis.Overexpression of GbRLK may improve stress tolerance by regulating stress-responsive genes to reduce water loss.Further studying of GbRLK will help elucidate abiotic stress signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Key Laboratory of Crop Genetics & Germplasm Enhancement, MOE Hybrid Cotton R&D Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China.

ABSTRACT

Background: Cotton (Gossypium spp.) is widely cultivated due to the important economic value of its fiber. However, extreme environmental degradation impedes cotton growth and production. Receptor-like kinase (RLK) proteins play important roles in signal transduction and participate in a diverse range of processes in response to plant hormones and environmental cues. Here, we introduced an RLK gene (GbRLK) from cotton into Arabidopsis and investigated its role in imparting abiotic stress tolerance.

Results: GbRLK transcription was induced by exogenously supplied abscisic acid (ABA), salicylic acid, methyl jasmonate, mock drought conditions and high salinity. We cloned the promoter sequence of this gene via self-formed adaptor PCR. Sequence analysis revealed that the promoter region contains many cis-acting stress-responsive elements such as ABRE, W-Box, MYB-core, W-Box core, TCA-element and others. We constructed a vector containing a 1,890-bp sequence in the 5' region upstream of the initiation codon of this promoter and transformed it into Arabidopsis thaliana. GUS histochemical staining analysis showed that GbRLK was expressed mainly in leaf veins, petioles and roots of transgenic Arabidopsis, but not in the cotyledons or root hairs. GbRLK promoter activity was induced by ABA, PEG, NaCl and Verticillium dahliae. Transgenic Arabidopsis with constitutive overexpression of GbRLK exhibited a reduced rate of water loss in leaves in vitro, along with improved salinity and drought tolerance and increased sensitivity to ABA compared with non-transgenic Col-0 Arabidopsis. Expression analysis of stress-responsive genes in GbRLK Arabidopsis revealed that there was increased expression of genes involved in the ABA-dependent signaling pathway (AtRD20, AtRD22 and AtRD26) and antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1) but not ion transporter genes (AtNHX1, AtSOS1).

Conclusions: GbRLK is involved in the drought and high salinity stresses pathway by activating or participating in the ABA signaling pathway. Overexpression of GbRLK may improve stress tolerance by regulating stress-responsive genes to reduce water loss. GbRLK may be employed in the genetic engineering of novel cotton cultivars in the future. Further studying of GbRLK will help elucidate abiotic stress signaling pathways.

Show MeSH
Related in: MedlinePlus