35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress

Kiselev, Konstantin V.; Aleynova, Olga A.; Ogneva, Zlata V.; Suprun, Andrey R.; Dubrovina, Alexandra S.

doi:10.1007/s11033-021-06235-x

Cited by 20 publications

(22 citation statements)

References 24 publications

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“…The 35S promoter is the most frequently used promoter, with a strong and constitutive expression pattern [ 46 ]. Although it functions as a constitutive expression pattern, the 35S promoter has variable expression patterns in different organs of Arabidopsis thaliana and in response to abiotic stress [ 46 , 47 ]. However, the phenomena reported in our study on transgenic lines, namely that salt stress induced a sharp miRNA-mediated reduction in the expression of SmAP2-17 , has not been previously reported.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of the Salix matsudana SmAP2-17 gene improves Arabidopsis salinity tolerance by enhancing the expression of SOS3 and ABI5

Chen

Dai

et al. 2022

BMC Plant Biol

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Background Salix matsudana (Koidz.) is a widely planted ornamental allotetraploid tree species. Genetic engineering can be used to enhance the tolerance of this species to soil salinization, endowing varieties with the ability to grow along coastlines, thereby mitigating afforestation and protecting the environment. The AP2/ERF family of transcription factors (TFs) plays multidimensional roles in plant biotic/abiotic stress tolerance and plant development. In this study, we cloned the SmAP2-17 gene and performed functional analysis of its role in salt tolerance. This study aims to identify key genes for future breeding of stress-resistant varieties of Salix matsudana. Results SmAP2-17 was predicted to be a homolog of AP2-like ethylene-responsive transcription factor ANT isoform X2 from Arabidopsis, with a predicted ORF of 2058 bp encoding an estimated protein of 685 amino acids containing two conserved AP2 domains (PF00847.20). SmAP2-17 had a constitutive expression pattern and was localized to the nucleus. The overexpression of the native SmAP2-17 CDS sequence in Arabidopsis did not increase salt tolerance because of the reduced expression level of ectopic SmAP2-17, potentially caused by salt-induced RNAi. Transgenic lines with high expression of optimized SmAP2-17 CDS under salt stress showed enhanced tolerance to salt. Moreover, the expression of general stress marker genes and important salt stress signaling genes, including RD29A, ABI5, SOS3, AtHKT1, and RBohF, were upregulated in SmAP2-17-overexpressed lines, with expression levels consistent with that of SmAP2-17 or optimized SmAP2-17. Promoter activity analysis using dual luciferase analysis showed that SmAP2-17 could bind the promoters of SOS3 and ABI5 to activate their expression, which plays a key role in regulating salt tolerance. Conclusions The SmAP2-17 gene isolated from Salix matsudana (Koidz.) is a positive regulator that improves the resistance of transgenic plants to salt stress by upregulating SOS3 and ABI5 genes. This study provides a potential functional gene resource for future generation of salt-resistant Salix lines by genetic engineering.

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Section: Discussionmentioning

confidence: 99%

Overexpression of the Salix matsudana SmAP2-17 gene improves Arabidopsis salinity tolerance by enhancing the expression of SOS3 and ABI5

Chen

Dai

et al. 2022

BMC Plant Biol

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“…To verify that the transgene suppressing effect induced by exogenous dsRNAs is sequence-specific, we used two transgenic Arabidopsis lines KA0-1 and KA0-2, bearing the NPTII transgene under the control of the doubled cauliflower mosaic virus (CaMV) 35S promoter ( Figure 1 a). The KA0-1 and KA0-2 lines have previously been obtained and show high levels of NPTII transgene expression [ 27 , 42 , 43 ]. A large fragment of the NPTII gene (599 bp out of 798 bp) and the complete EGFP coding region (720 bp) was amplified by PCR for further in vitro transcription and dsRNA production ( Figure 1 a,b).…”

Section: Resultsmentioning

confidence: 99%

The Specificity of Transgene Suppression in Plants by Exogenous dsRNA

Kiselev

Suprun

Aleynova

et al. 2022

Plants

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The phenomenon of RNA interference (RNAi) is widely used to develop new approaches for crop improvement and plant protection. Recent investigations show that it is possible to downregulate plant transgenes, as more prone sequences to silencing than endogenous genes, by exogenous application of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs). However, there are scarce data on the specificity of exogenous RNAs. In this study, we explored whether plant transgene suppression is sequence-specific to exogenous dsRNAs and whether similar effects can be caused by exogenous DNAs that are known to be perceived by plants and induce certain epigenetic and biochemical changes. We treated transgenic plants of Arabidopsis thaliana bearing the neomycin phosphotransferase II (NPTII) transgene with specific synthetic NPTII-dsRNAs and non-specific dsRNAs, encoding enhanced green fluorescent protein (EGFP), as well as with DNA molecules mimicking the applied RNAs. None of the EGFP-dsRNA doses resulted in a significant decrease in NPTII transgene expression in the NPTII-transgenic plants, while the specific NPTII-dsRNA significantly reduced NPTII expression in a dose-dependent manner. Long DNAs mimicking dsRNAs and short DNA oligonucleotides mimicking siRNAs did not exhibit a significant effect on NPTII transgene expression. Thus, exogenous NPTII-dsRNAs induced a sequence-specific and RNA-specific transgene-suppressing effect, supporting external application of dsRNAs as a promising strategy for plant gene regulation.

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“…To demonstrate that exogenous genes can be transiently expressed in seedlings, the eGFP gene was selected because it can acquire transient overexpression with the cauliflower mosaic virus (CaMV) 35S promoter [ 30 ]. The plant binary expression vector pART27-eGFP (College of Agriculture, Yangtze University) was transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-melt method [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

An efficient overexpression method for studying genes in Ricinus that transport vectorized agrochemicals

Xiao

Zhang

et al. 2022

Plant Methods

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Background Plant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals. Therefore, transporters associated with phloem loading of vectorized agrochemicals have drawn increasing attention. As a model system, castor bean (Ricinus communis L.) has been widely used to detect the phloem mobility of agrochemicals. However, there is still a lack of an efficient protocol for the Ricinus seedling model system that can be directly used to investigate the recognition and phloem loading functions of plasmalemma transporters toward vectorized agrochemicals. Results Here, using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Strong fluorescence signals were observed in leaves, demonstrating that exogenous genes can be successfully overexpressed in seedlings. Subsequently, gene expression time and vacuum infiltration parameters were optimized. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Parameter optimization showed Agrobacterium concentration at OD600 = 1.2, and infiltration for 20 min (0.09 MPa), return to atmospheric pressure, and then infiltration for another 20 min, were the suitable transformation conditions. To test the application of vacuum agroinfiltration in directly examining the loading functions of plasma membrane transporters to vectorized agrochemicals in seedlings, two LHT (lysine/histidine transporter) genes, RcLHT1 and RcLHT7, were overexpressed. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. Furthermore, the concentrations of l-Val-PCA (l-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of l-Val-PCA. Conclusions We successfully constructed a transient expression system in Ricinus seedlings and laid the foundation for researchers to directly investigate the loading functions of plasma membrane transporters to vectorized agrochemicals in the Ricinus system.

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35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress

Cited by 20 publications

References 24 publications

Overexpression of the Salix matsudana SmAP2-17 gene improves Arabidopsis salinity tolerance by enhancing the expression of SOS3 and ABI5

Overexpression of the Salix matsudana SmAP2-17 gene improves Arabidopsis salinity tolerance by enhancing the expression of SOS3 and ABI5

The Specificity of Transgene Suppression in Plants by Exogenous dsRNA

An efficient overexpression method for studying genes in Ricinus that transport vectorized agrochemicals

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