Abstract
In response to water-deficit stress, plants alter expression of thousands of genes and as a result, cellular, physiological, and biochemical processes are modified. Understanding the functional role of water-deficit stress-responsive genes is important in order to develop stress-tolerant plants. RNA interference (RNAi) technology is one of the potential reverse genetics tool for assessing the functional significance of these genes. We describe here the protocols for developing stable gene knockdown lines for stress-induced genes using RNAi. In addition, stress imposition method that allows plants to experience gradual acclimation stress is enumerated. Further, precautions that should be taken while developing RNAi lines and during stress imposition are discussed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ramanjulu, S. and Bartels, D. (2002) Drought- and desiccation-induced modulation of gene expression in plants. Plant Cell Environ 25, 141–151.
Seki, M., Kamei, A., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2003) Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol 14, 194–199.
Small, I. (2007) RNAi for revealing and engineering plant gene functions. Curr Opin Biotechnol 18, 148–153.
Matthew, L. (2004) RNAi for plant functional genomics. Compar Funct Genom 5, 240–244.
Travella, S., Klimm, T.E., and Keller, B. (2006) RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat. Plant Physiol 142, 6–20.
Senthil-Kumar, M., Ramegowda, H.V., Hema, R., Mysore, K.S., and Udayakumar, M. (2008) Virus-induced gene silencing and its application in characterizing genes involved in water deficit stress tolerance. J Plant Physiol 165, 1404–1421.
Senthil-Kumar, M. (2007) Functional characterization of peanut water deficit stress-induced genes: an approach based on virus-induced gene silencing (VIGS) and RNAi [PhD thesis]. Bangalore: University of Agricultural Sciences.
Mittler, R., Kim, Y., Song, L., Coutu, J., Coutu, A., Ciftci-Yilmaz, S., Lee, H., Stevenson, B., and Zhu, J.-K. (2006) Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS Lett 580, 6537–6542.
Tateishi, Y., Nakagawa, T., and Esaka, M. (2005) Osmotolerance and growth stimulation of transgenic tobacco cells accumulating free proline by silencing proline dehydrogenase expression with double-stranded RNA interference technique. Physiol Plant 125, 224–234.
Yang Wang, J.Y., Kuzma, M., Chalifoux, M., Sample, A., McArthur, C., Uchacz, T., Sarvas, C., Wan, J., Dennis, D.T., McCourt, P., and Huang, Y. (2005) Molecular tailoring of farnesylation for plant drought tolerance and yield protection. Plant J 43, 413–424.
Verslues, P.E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., and Zhu, J.K. (2006) Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J 45, 523–539.
Jones, H.G. (2007) Monitoring plant and soil water status: established and novel methods revisited and their relevance to studies of drought tolerance. J Exp Bot 58, 119–130.
Senthil-Kumar, M., Kumar, G., Srikanthbabu, V., and Udayakumar, M. (2007) Assessment of variability in acquired thermotolerance: potential option to study genotypic response and the relevance of stress genes. J Plant Physiol 164, 111–125.
Travella, S. and Keller, B. (2009) Down-regulation of gene expression by RNA-induced gene silencing. In Transgenic Wheat, Barley and Oats, Volume 478 (Jones, H.D. and Shewry, P.R. eds.). Methods in Molecular Biology, Humana Press, Springer, New York, pp. 185–199.
Karimi, M., Inze, D., and Depicker, A. (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7, 193–195.
Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, 45.
Towill, L.E. and Mazur, P. (1975) Studies on the reduction of 2,3,5-triphenyl tetrazolium chloride as a viability assay for plant tissue cultures. Can J Bot 53, 1097–1102.
Tripathy, J.N., Zhang, J., Robin, S., Nguyen, T.T., and Nguyen, H.T. (2000) QTLs for cell-membrane stability mapped in rice (Oryza sativa L.) under drought stress. Theor Appl Genet 100, 1197–2202.
Xu, P., Zhang, Y., Kang, L., Roossinck, M.J., and Mysore, K.S. (2006) Computational estimation and experimental verification of off-target silencing during post-transcriptional gene silencing in plants. Plant Physiol 142, 429–440.
Helliwell, C.A. and Waterhouse, P.M. (2005) Constructs and methods for hairpin RNA-mediated gene silencing in plants. In Methods Enzymol, Volume 392 ed. (Engelke, D.R. and Rossi, J.J., eds.). Academic Press, pp. 24–35.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press
About this protocol
Cite this protocol
Senthil-Kumar, M., Udayakumar, M., Mysore, K.S. (2010). Functional Characterization of Water-Deficit Stress Responsive Genes Using RNAi. In: Sunkar, R. (eds) Plant Stress Tolerance. Methods in Molecular Biology, vol 639. Humana Press. https://doi.org/10.1007/978-1-60761-702-0_11
Download citation
DOI: https://doi.org/10.1007/978-1-60761-702-0_11
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-701-3
Online ISBN: 978-1-60761-702-0
eBook Packages: Springer Protocols