Abstract
In this study, we developed a simple and efficient transient transformation system, which can be used in homologous expression or reverse genetic study of the plants. A system for characterizing gene function in response to stress tolerance was also developed based on this transformation method. The overexpression and RNAi-silencing of a bZIP gene from Tamarix hispida, ThbZIP1, were performed in T. hispida using this transformation method. Real-time PCR showed that the expression of ThbZIP1 was highly up- and down-regulated in the plants with overexpression and RNAi-silenced expression of ThbZIP1, respectively, when compared with control plants (transiently transformed with empty pROK2). A physiological study showed that ThbZIP1 can enhance the activities of both peroxidase (POD) and superoxide dismutase (SOD), and decrease electrolyte leakage rate and levels of reactive oxygen species (ROS) and malondialdehyde (MDA) under salt stress conditions. Furthermore, ThbZIP1 is found to mediate stress tolerance by regulating the expression of SOD and POD genes. These results suggested that this transient transformation system is an effective method for determining the function of a gene in response to abiotic stress in plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116
Chen X, Equi R, Baxter H, Berk K, Han J, Agarwal S, Zale J (2010) A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings. Biotechnol Biofuels 3:9
Dal Santo S, Stampfl H, Krasensky J, Kempa S, Gibon Y, Petutschnig E, Rozhon W, Heuck A, Clausen T, Jonak C (2012) Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in arabidopsis. Plant Cell 24:3380–33992
Gao CQ, Wang YC, Liu GF, Wang C, Jiang J, Yang CP (2010) Cloning of ten peroxidase (POD) genes from Tamarix hispida and characterization of their responses to abiotic stress. Plant Mol Biol Rep 28:77–89
Goodin MM, Dietzgen RG, Schichnes D, Ruzin S, Jackson AO (2002) pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J 31:375–383
Jefferson RA (1989) The GUS reporter gene system. Nature 342:837–838
Kapila J, De Rycke R, Van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108
Kim M, Ahn JW, Jin UH, Choi D, Paek KH, Pai HS (2003) Activation of the programmed cell death pathway by inhibition of proteasome function in plants. J Biol Chem 278:19406–19415
Kirienko DR, Luo A, Sylvester AW (2012) Reliable transient transformation of intact maize leaf cells for functional genomics and experimental study. Plant Physiol 159:1309–1318
Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P (1988) Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Proc Natl Acad Sci USA 85(22):8502–8505
Koroleva OA, Tomlinson ML, Leader D, Shaw P, Doonan JH (2005) High throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J 41:162–174
Kuttenkeuler D, Boutros M (2004) Genome-wide RNAi as a route to gene function in Drosophila. Brief Funct Genomic Proteomic 3(2):168–176
Li HY, Wang YC, Jiang J, Liu GF, Gao CQ, Yang CP (2009a) Identification of genes responsive to salt stress on Tamarix hispida roots. Gene 433:65–71
Li J, Park E, von Arnim AG, Nebenführ A (2009b) The FAST technique: a simplified Agrobacteriumbased transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 (−Delta Delta C(T)) Method. Methods 25:402–408
Lu Y, Chen X, Wu Y, Wang Y, He Y, Wu Y (2013) Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay. PLoS ONE 8:2
Phillipson JD (2001) Phytochemistry and medicinal plants. Phytochemistry 56:237–243
Sheludko YV, Sindarovska YR, Gerasymenko IM, Bannikova MA, Kuchuk NV (2006) Comparison of several Nicotiana species as host for high-scale Agrobacterium-mediated transient expression. Biotechnol Bioeng 96:608–614
Simmons CW, VanderGheynst JS, Upadhyaya SK (2009) A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnol Bioeng 102:965–970
Takata N, Eriksson ME (2012) A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides). Plant Methods 8:30
Van der Hoorn RA, Laurent F, Roth R, De Wit PJ (2000) Agroinfiltration is a versatile tool that facilitates comparative analysis of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Mol Plant Microbe Interact 13:439–446
VanderGheynst JS, Guo HY, Simmons CW (2008) Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass (Panicum virgatum). Biomass Bioenergy 32:372–379
Wang YC, Gao CQ, Liang YN, Wang C, Yang CP, Liu GF (2010) A novel bZIP gene from Tamarix hispida mediates physiological responses to salt stress in tobacco plants. J Plant Physiol 167:222–230
Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato, and Arabidopsis. Plant Biotechnol J 3:259–273
Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572
Zhang X, Wang L, Meng H, Wen H, Fan Y, Zhao J (2011) Maize ABP9 enhances tolerance to multiple stresses in transgenic Arabidopsis by modulating ABA signaling and cellular levels of reactive oxygen species. Plant Mol Biol 75:365–378
Zheng L, Liu GF, Meng XN, Li YB, Wang YC (2012) A versatile Agrobacterium-mediated transient gene expression system for herbaceous plants and trees. Biochem Genet 50:761–769
Acknowledgments
This work was supported by The Hundred Talents Program of the Chinese Academy of Sciences, and Foundation for the Author of National Excellent Doctoral Dissertation of China (No.200973).
Author Contributions Statement
Y.C. Wang and X.Y. Ji conceived the project and designed experiments. X.Y. Ji, L. Zheng, Y.J. Liu, X.G. Nie and S.N. Liu participated in the experiments. X.Y. Ji performed the data analysis. X.Y. Ji, and Y.C. Wang drafted the manuscript. Y.C. Wang provided reagents and analysis tools.
Conflict of Interest Disclosure
The authors declare that they have no conflict of interest
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(DOC 53 kb)
Rights and permissions
About this article
Cite this article
Ji, X., Zheng, L., Liu, Y. et al. A Transient Transformation System for the Functional Characterization of Genes Involved in Stress Response. Plant Mol Biol Rep 32, 732–739 (2014). https://doi.org/10.1007/s11105-013-0683-z
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11105-013-0683-z