Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 119, Issue 1, pp 211–219 | Cite as

A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes

  • Yu Bai
  • Ning Han
  • Jinxia Wu
  • Yinong Yang
  • Junhui Wang
  • Muyuan Zhu
  • Hongwu Bian
Original Paper

Abstract

Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.

Keywords

Barley Protoplast isolation PEG-mediated transfection MicroRNAs MicroRNA target genes 

References

  1. Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2:13. doi:10.1186/1746-4811-2-13 PubMedCrossRefPubMedCentralGoogle Scholar
  2. Bian H, Xie Y, Guo F, Han N, Ma S, Zeng Z, Wang J, Yang Y, Zhu M (2012) Distinctive expression patterns and roles of the miRNA393/TIR1 homolog module in regulating flag leaf inclination and primary and crown root growth in rice (Oryza sativa). New Phytol 196(1):149–161. doi:10.1111/j.1469-8137.2012.04248.x PubMedCrossRefGoogle Scholar
  3. Cahoon EB, Hall SE, Ripp KG, Ganzke TS, Hitz WD, Coughlan SJ (2003) Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat Biotechnol 21(9):1082–1087. doi:10.1038/nbt853 PubMedCrossRefGoogle Scholar
  4. Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice. Mol Plant Pathol 7(5):417–427. doi:10.1111/j.1364-3703.2006.00346.x PubMedCrossRefGoogle Scholar
  5. Colaiacovo M, Subacchi A, Bagnaresi P, Lamontanara A, Cattivelli L, Faccioli P (2010) A computational-based update on microRNAs and their targets in barley (Hordeum vulgare L.). BMC Genom 11:595. doi:10.1186/1471-2164-11-595 CrossRefGoogle Scholar
  6. Faraco M, Di Sansebastiano GP, Spelt K, Koes RE, Quattrocchio FM (2011) One protoplast is not the other! Plant Physiol 156(2):474–478. doi:10.1104/pp.111.173708 PubMedCrossRefPubMedCentralGoogle Scholar
  7. Fischer R, Hain R (1995) Tobacco protoplast transformation and use for functional analysis of newly isolated genes and gene constructs. Methods Cell Biol 50:401–410PubMedCrossRefGoogle Scholar
  8. Garcia I, Rodgers M, Lenne C, Rolland A, Sailland A, Matringe M (1997) Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA. Biochem J 325(Pt 3):761–769PubMedPubMedCentralGoogle Scholar
  9. Garcia I, Rodgers M, Pepin R, Hsieh TF, Matringe M (1999) Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco. Plant Physiol 119(4):1507–1516PubMedCrossRefPubMedCentralGoogle Scholar
  10. Gudesblat GE, Schneider-Pizon J, Betti C, Mayerhofer J, Vanhoutte I, van Dongen W, Boeren S, Zhiponova M, de Vries S, Jonak C, Russinova E (2012) SPEECHLESS integrates brassinosteroid and stomata signalling pathways. Nat Cell Biol 14(5):548–554. doi:10.1038/ncb2471 PubMedCrossRefGoogle Scholar
  11. Holm PB, Olsen O, Schnorf M, Brinch-Pedersen H, Knudsen S (2000) Transformation of barley by microinjection into isolated zygote protoplasts. Transgenic Res 9(1):21–32PubMedCrossRefGoogle Scholar
  12. Hooley R (1982) Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid. Planta 154(1):29–40. doi:10.1007/bf00385493 PubMedCrossRefGoogle Scholar
  13. Kim J, Somers DE (2010) Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts. Plant Physiol 154(2):611–621. doi:10.1104/pp.110.162271 PubMedCrossRefPubMedCentralGoogle Scholar
  14. Li JF, Chung HS, Niu Y, Bush J, McCormack M, Sheen J (2013) Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25(5):1507–1522. doi:10.1105/tpc.113.112235 PubMedCrossRefPubMedCentralGoogle Scholar
  15. Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington JC (2007) Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. Plant J 52(1):133–146. doi:10.1111/j.1365-313X.2007.03218.x PubMedCrossRefGoogle Scholar
  16. Lynn K, Fernandez A, Aida M, Sedbrook J, Tasaka M, Masson P, Barton MK (1999) The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 126(3):469–481PubMedGoogle Scholar
  17. Macovei A, Tuteja N (2012) microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.). BMC Plant Biol 12:183. doi:10.1186/1471-2229-12-183 PubMedCrossRefPubMedCentralGoogle Scholar
  18. Martinoia Enrico, Heck Urs, Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves. Nature 289:292–294. doi:10.1038/289292a0 CrossRefGoogle Scholar
  19. Mayer KF, Waugh R, Brown JW, Schulman A, Langridge P, Platzer M, Fincher GB, Muehlbauer GJ, Sato K, Close TJ, Wise RP, Stein N (2012) A physical, genetic and functional sequence assembly of the barley genome. Nature 491(7426):711–716. doi:10.1038/nature11543 PubMedGoogle Scholar
  20. Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104(1):34–41. doi:10.1263/jbb.104.34 PubMedCrossRefGoogle Scholar
  21. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312(5772):436–439. doi:10.1126/science.1126088 PubMedCrossRefGoogle Scholar
  22. Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51(6):1126–1136. doi:10.1111/j.1365-313X.2007.03212.x PubMedCrossRefGoogle Scholar
  23. Nevo E (2013) Evolution of wild barley and barley improvement. In: Zhang G, Li C, Liu X (eds) Advance in barley sciences. Springer Netherlands, pp 1–23. doi:10.1007/978-94-007-4682-4_1
  24. Okuno T, Furusawa I (1977) A simple method for the isolation of intact mesophyll protoplasts from cereal plants. Plant Cell Physiol 18(6):1357–1362Google Scholar
  25. Omirulleh S, Abraham M, Golovkin M, Stefanov I, Karabaev MK, Mustardy L, Morocz S, Dudits D (1993) Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize. Plant Mol Biol 21(3):415–428PubMedCrossRefGoogle Scholar
  26. Salmenkallio-Marttila M, Aspegren K, Akerman S, Kurtén U, Mannonen L, Ritala A, Teeri TH, Kauppinen V (1995) Transgenic barley (Hordeum vulgate L.) by electroporation of protoplasts. Plant Cell Rep 15:301–304. doi:10.1007/BF00193741 PubMedGoogle Scholar
  27. Schauer SE, Golden TA, Merchant DS, Patra BN, Lang JD, Ray S, Chakravarti B, Chakravarti DN, Ray A (2013) DCL1, a protein that produces plant microRNA, coordinates meristem activity. bioRxiv. http://biorxiv.org/content/early/2013/12/16/001438. doi:10.1101/001438
  28. Schutze K, Harter K, Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein–protein interactions in living plant cells. Methods Mol Biol 479:189–202. doi:10.1007/978-1-59745-289-2_12 PubMedCrossRefGoogle Scholar
  29. Seguin A, Lalonde M (1988) Gene transfer by electroporation in Betulaceae protoplasts: Alnus incana. Plant Cell Rep 7(6):367–370. doi:10.1007/BF00269514 PubMedGoogle Scholar
  30. Shi Y, Evans JE, Rock KL (2003) Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425(6957):516–521PubMedCrossRefGoogle Scholar
  31. Stanca A, Tumino G, Pagani D, Rizza F, Alberici R, Lundqvist U, Morcia C, Tondelli A, Terzi V (2013) The “Italian” barley genetic mutant collection: conservation, development of new mutants and use. In: Zhang G, Li C, Liu X (eds) Advance in barley sciences. Springer Netherlands, pp 47–56. doi:10.1007/978-94-007-4682-4_4
  32. Tan B, Xu M, Chen Y, Huang M (2013) Transient expression for functional gene analysis using Populus protoplasts. Plant Cell Tissue Organ Cult 114(1):11–18. doi:10.1007/s11240-013-0299-x CrossRefGoogle Scholar
  33. Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40(3):428–438. doi:10.1111/j.1365-313X.2004.02219.x PubMedCrossRefGoogle Scholar
  34. Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104(1):37–48. doi:10.1104/pp.104.1.37 PubMedPubMedCentralGoogle Scholar
  35. Wehner N, Hartmann L, Ehlert A, Bottner S, Onate-Sanchez L, Droge-Laser W (2011) High-throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function. Plant J 68(3):560–569. doi:10.1111/j.1365-313X.2011.04704.x PubMedCrossRefGoogle Scholar
  36. Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6(6):1975–1983. doi:10.1093/mp/sst119 PubMedCrossRefGoogle Scholar
  37. Yang Y, Jin H, Chen Y, Lin W, Wang C, Chen Z, Han N, Bian H, Zhu M, Wang J (2012) A chloroplast envelope membrane protein containing a putative LrgB domain related to the control of bacterial death and lysis is required for chloroplast development in Arabidopsis thaliana. New Phytol 193(1):81–95. doi:10.1111/j.1469-8137.2011.03867.x PubMedCrossRefGoogle Scholar
  38. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7):1565–1572. doi:10.1038/nprot.2007.199 PubMedCrossRefGoogle Scholar
  39. You Y, Shirako Y (2013) Evaluation of host resistance to barley yellow mosaic virus infection at the cellular and whole-plant levels. Plant Pathol 62(1):226–232. doi:10.1111/j.1365-3059.2012.02616.x CrossRefGoogle Scholar
  40. Zhang H, Li L (2013) SQUAMOSA promoter binding protein-like7 regulated microRNA408 is required for vegetative development in Arabidopsis. Plant J 74(1):98–109. doi:10.1111/tpj.12107 PubMedCrossRefGoogle Scholar
  41. Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7(1):30. doi:10.1186/1746-4811-7-30 PubMedCrossRefPubMedCentralGoogle Scholar
  42. Zhou M, Gu L, Li P, Song X, Wei L, Chen Z, Cao X (2010) Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica). Front Biol 5(1):67–90. doi:10.1007/s11515-010-0007-8 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Yu Bai
    • 1
  • Ning Han
    • 1
  • Jinxia Wu
    • 1
  • Yinong Yang
    • 1
    • 2
  • Junhui Wang
    • 1
  • Muyuan Zhu
    • 1
  • Hongwu Bian
    • 1
  1. 1.Institute of Genetics, College of Life SciencesZhejiang UniversityHangzhouChina
  2. 2.Department of Plant Pathology and Environmental Microbiology, and Huck Institutes of Life SciencesPennsylvania State UniversityUniversity ParkUSA

Personalised recommendations