Factors Influencing the Preparation of Wheat Mesophyll Protoplasts and the Application of Wheat Mesophyll Protoplasts in Gene Transient Expression

doi:10.7668/hbnxb.2015.06.008

Abstract

Abstract: In order to establish a stable and efficient protocol for the isolation of wheat mesophyll protoplasts,experiments of orthogonal and completely random designs were conducted to analyze the factors influencing the yield of mesophyll protoplasts from wheat seedling leaves.It was found that the mannitol concentration in the enzyme solution showed the greatest impact on protoplast yield,followed by the time of enzymatic treatment,the cellulase concentration and the macerozyme in the enzyme solution.The mannitol concentration and the cellulase concentration showed a mutual promotion effect on the yield of protoplast.If a prolonged time of enzymatic treatment was applied,low concentrations of cellulase released more round-shaped protoplasts and sustained a more stable quantity of protoplasts after the time of the peak yield value than high concentrations of cellulase.Other factors influencing protoplast yield include seedling age,light conditions and the position of leaves collected for protoplast isolation.Based on these results,an optimized set of parameters for wheat mesophyll protoplast isolation was determined,in which the second leaves of 10-day-old wheat seedlings grown under dark conditions were selected as the explants and were digested with an enzyme solution containing 0.5% cellulose,0.6% macerozyme and 0.5 mol/L mannitol for 6 hours.Uniform,round-shaped protoplasts were prepared by using this set of parameters and a yield up to (7.28±2.18)×10⁶ per gram leaf fresh weight could be obtained.The percentage of viable protoplasts was up to (95.06±2.66)%.The PEG-Ca²⁺-mediated transformation procedure was applied to transform gene constructs into prepared mesophyll protoplasts and a transformation efficiency of (61.31±5.74)% was determined by using GFP as a detecting marker.The GFP or DsRed fusion protein of wheat TaATG8h and Arabidopsis AtPIP2 ; 1 ,when expressed wheat mesophyll protoplasts,showed correct subcellular localization.

Key words: Wheat, Protoplast, Gene transformation, Transient expression

CLC Number:

S512
Q819

DU Xiao-min, WANG Jun, AN Zi-xuan, PEI Dan, WANG Hua-zhong. Factors Influencing the Preparation of Wheat Mesophyll Protoplasts and the Application of Wheat Mesophyll Protoplasts in Gene Transient Expression[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(6): 52-60. doi: 10.7668/hbnxb.2015.06.008.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2015.06.008

http://www.hbnxb.net/EN/Y2015/V30/I6/52

References

[1] Seguin A,Lalonde M.Gene transfer by electroporation in Betulaceae protoplasts:Alnus incana[J].Plant Cell Reports,1988,7(6):367-370.
[2] Salmenkallio-Marttila M,Aspegren K,Akerman S,et al.Transgenic barley (Hordeum vulgare L.) by electroporation of protoplasts[J].Plant Cell Reports,1995,15(3-4):301-304.
[3] Holm P B,Olsen O,Schnorf M,et al.Transformation of barley by microinjection into isolated zygote protoplasts[J].Transgenic Research,2000,9(1):21-32.
[4] Yoo S D,Cho Y H,Sheen J.Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis[J].Nature Protocols,2007,2(7):1565-1572.
[5] Sheen J.Signal transduction in maize and Arabidopsis mesophyll protoplasts[J].Plant Physiology,2001,127(4):1466-1475.
[6] Chen S,Tao L,Zeng L,et al.A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice[J].Molecular Plant Pathology,2006,7(5):417-427.
[7] Omirulleh S,Abraham M,Golovkin M,et al.Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize[J].Plant Molecular Biology,1993,21(3):415-428.
[8] Fischer R,Hain R.Tobacco protoplast transformation and use for functional analysis of newly isolated genes and gene constructs[J].Methods in Cell Biology,1995,50:401-410.
[9] Tan B,Xu M,Chen Y,et al.Transient expression for functional gene analysis using Populus protoplasts[J].Plant Cell,Tissue and Organ Culture,2013,114(1):11-18.
[10] Bai Y,Han N,Wu J X,et al.A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes[J].Plant Cell,Tissue and Organ Culture,2014,119:211-219.
[11] Faraco M,Di Sansebastiano G P,Spelt K,et al.One protoplast is not the other[J].Plant Physiology,2011,156(2):474-478.
[12] Park C J,Bart R,Chern M,et al.Overexpression of the endoplasmic reticulum chaperone BiP3 regulates XA21-mediated innate immunity in rice[J].PLoS One,2010,5(2):9262.
[13] Schutze K,Harter K,Chaban C.Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells[J].Methods in Molecular Biology,2009,479:189-202.
[14] Walter M,Chaban C,Schütze K,et al.Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation[J].The Plant Journal,2004,40(3):428-438.
[15] Shi Y,Evans J E,Rock K L.Molecular identification of a danger signal that alerts the immune system to dying cells[J].Nature,2003,425(6957):516-521.
[16] Im J H,Yoo S D.Transient expression in Arabidopsis leaf mesophyll protoplast system for cell-based functional analysis of MAPK cascades signaling[J].Methods in Molecular Biology,2014,1171:3-12.
[17] Fraiture M,Zheng X,Brunner F.An Arabidopsis and tomato mesophyll protoplast system for fast identification of early MAMP-triggered immunity-suppressing effectors[J].Methods in Molecular Biology,2014,1127:213-230.
[18] Wehner N,Hartmann L,Ehlert A,et al.High-throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function[J].The Plant Journal,2011,68(3):560-569.
[19] Sahoo D K,Sarkar S,Raha S,et al.Comparative analysis of synthetic DNA promoters for high-level gene expression in plants[J].Planta,2014,240(4):855-875.
[20] Xie K,Yang Y.RNA-guided genome editing in plants using a CRISPR-Cas system[J].Molecular Plant,2013,6(6):1975-1983.
[21] Li J F,Chung H S,Niu Y,et al.Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants[J].The Plant Cell,2013,25(5):1507-1522.
[22] Bart R,Chern M,Park C J,et al.A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts[J].Plant Methods,2006,2(1):13.
[23] Shan Q,Wang Y,Li J,et al.Genome editing in rice and wheat using the CRISPR Cas system[J].Nature Protocols,2014,9(10):2395-2410.
[24] Liu D,Li A,Mao X,et al.Cloning and characterization of TaPP2AbB"-α,a member of the PP2A regulatory subunit in wheat[J].PLoS One,2014,9(4):94430.
[25] Yang K,Rong W,Qi L,et al.Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis[J].Scientific Reports,2013(3):3021.
[26] Zhang L,Zhang L,Xia C,et al.A novel wheat bZIP transcription factor,TabZIP60,confers multiple abiotic stress tolerances in transgenic Arabidopsis[J].Physiologia Plantarum,2015,153(4):538-554.
[27] Xu D B,Gao S Q,Ma Y Z,et al.ABI-like transcription factor gene TaABL1 from wheat improves multiple abiotic stress tolerances in transgenic plants[J].Functional and Integrative Genomics,2014,14(4):717-730.
[28] 孙鹤,郎志宏,朱莉,等.玉米、小麦、水稻原生质体制备条件优化[J].生物工程学报,2013,29:224-234.
[29] 李妮娜,丁林云,张志远,等.棉花叶肉原生质体分离及目标基因瞬时表达体系的建立[J].作物学报,2014,40(2):231-239.
[30] Evans P K,Keates A G,Cocking E C.Isolation of protoplasts from cereal leaves[J].Planta,1972,104(2):178-181.
[31] Huber S C,Edwards G E.An evaluation of some parameters required for the enzymatic isolation of cells and and protoplasts with CO₂ fixation capacity from C3 and C4 grasses[J].Physiologia Plantarum,1975,35(3):203-209.
[32] Sarhan F,Cesar D.High yield isolation of mesophyll protoplasts from wheat,barley and rye[J].Physiologia Plantarum,1988,72(2):337-342.
[33] Pei D,Zhang W,Sun H,et al.Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses[J].Plant Cell Reports,2014,33(10):1697-1710.
[34] Prak S,Hem S,Boudet J,et al.Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins:role in subcellular trafficking of AtPIP2;1 in response to salt stress[J].Molecular and Cellular Proteomics,2008,7(6):1019-1030.
[35] Jiang F,Zhu J,Liu H L.Protoplasts:A useful research system for plant cell biology,especially dedifferentiation[J].Protoplasma,2013,250(6):1231-1238.
[36] Edwards G E,Lee S S,Chen T M,et al.Carboxylation reactions and photosynthesis of carbon compounds in isolated mesophyll and bundle sheath cells of Digitaria sanguinalis (L.) Scop[J].Biochemical and Biophysical Research Communications,1970,39:389-395.
[37] Kanai R,Edwards G E.Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies[J].Plant Physiology,1973,51(6):1133-1137.
[38] Riazunnisa K,Lolla P,Scheibe R,et al.Preparation of Arabidopsis mesophyll protoplasts with high rates of photosynthesis[J].Physiologia Plantarum,2007,129(4):879-886.
[39] Zhang Y,Su J,Duan S,et al.A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes[J].Plant Methods,2011,7(1):30.
[40] Daveya M R,Anthonya P,Power J B,et al.Plant protoplasts:status and biotechnological perspectives[J].Biotechnology Advances,2005,23(2):131-171.
[41] Hooley R.Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid[J].Planta,1982,154(1):29-40.
[42] Krogaard H,Andersen A S.Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts[J].Physiologia Plantarum,1990,80(1):119-125.
[43] Pallardy S G,Kozlowski T T.Cuticle development in the stomatal region of Populus clones[J].New Phytologist,1980,85:363-368.

Please choose a citation manager

Content to export