Preparation of Leaf Mesophyll Protoplasts for Transient Gene Expression in Brachypodium distachyon

다국어 초록 (Multilingual Abstract)

Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and β-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion.

Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 μg of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence,the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular,molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.

참고문헌 (Reference)

1 Fischer R, "protoplast transformation and use for functional analysis of newly isolated genes and gene constructs" 50 : 401-410, 1995

2 Goodin MM, "pGD vectors: versatile tools for the expression of greenand red fluorescent protein fusions in agroinfiltrated plant leaves" 31 : 375-383, 2002

3 Jefferson RA, "fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants" 6 : 3901-3907, 1987

4 Manavella PA, "Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies" 4 : 1699-1707, 2009

5 Abel S, "Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression" 5 : 421-427, 1994

6 Hoffman A, "Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana" 36 : 53-58, 1994

7 Luehrsen KR, "Transient expression analysis in plants using firefly luciferase reporter gene" 216 : 397-414, 1992

8 Datta K, "Transformation of rice via PEG-mediated DNA uptake into protoplasts" 111 : 335-347, 1999

9 Widholm JM, "The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells" 47 : 189-194, 1972

10 Wagner GJ, "The plant protoplast: A useful tool for plant research and student instruction" 28 : 95-101, 1978

11 Rao AQ, "The myth of plant transformation" 27 : 753-763, 2009

12 Sheen J, "Signal transduction in maize and Arabidopsis mesophyll protoplasts" 127 : 1466-1475, 2001

13 Hansen G, "Recent advances in the transformation of plants" 4 : 226-231, 1999

14 Larkin PJ, "Purification and viability of plant protoplasts" 128 : 213-216, 1976

15 Mazarei M, "Protoplast isolation and transient gene expression in switchgrass" 3 : 354-359, 2008

16 Davey MR, "Protoplast applications in biotechnology, In Encyclopaedia of plant and crop science" Marcel Dekker 1061-1064, 2004

17 Davey MR, "Plant protoplasts: status and biotechnological perspectives" 23 : 131-171, 2005

18 Bruce WB, "Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment" 86 : 9692-9696, 1989

19 Loake GJ, "Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter" 3 : 829-840, 1991

20 Hong SY, "Phenological growth stages of Brachypodium distachyon: codification and description according to the BBCH scale" 51 : 612-620, 2011

21 Mathur J, "PEG-mediated protoplast transformation with naked DNA" 82 : 267-276, 1998

22 Rasmussen JO, "PEG mediated DNA uptake and transient GUS expression in carrot, rapeseed and soybean protoplasts" 89 : 199-207, 1993

23 Kim MJ, "Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis" 28 : 1159-1167, 2009

24 Liu CN, "Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens" 20 : 1071-1087, 1992

25 Filiz E, "Molecular, morphological, and cytological analysis of diverse Brachypodium distachyon inbred lines" 52 : 876-890, 2009

26 Datla RS, "Modified binary plant transformation vectors with the wild-type gene encoding NPTII" 122 : 383-384, 1992

27 Maas C, "Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts" 8 : 148-151, 1989

28 De Domenico S, "Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets" 53 : 858-868, 2011

29 Davey MR, "Isolation, culture, and plant regeneration from leaf protoplasts of Passiflora" 318 : 201-210, 2006

30 Power JB, "Isolation of leaf protoplasts: macromolecular uptake and growth substance response" 21 : 64-70, 1970

31 Koop HU, "Integration of foreign sequences into the tobacco plastomevia polyethylene glycol-mediated protoplast transformation" 199 : 193-201, 1996

32 Davey MR, "Higher plant protoplasts-retrospect and prospect" 16 : 219-299, 1983

33 Vogel J, "High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3" 27 : 471-478, 2008

34 Nurnberger T, "High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses" 78 : 449-460, 1994

35 Evans DA, "Handbook of Plant Cell Culture, In Handbook of Plant Cell Culture" MacMillan 124-176, 1983

36 Elliott AR, "Green fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells" 18 : 707-714, 1999

37 International Brachypodium Initiative, "Genome sequencing and analysis of the model grass Brachypodium distachyon" 463 : 763-768, 2010

38 Massa AN, "Gene space dynamics during the evolution of Aegilops tauschii, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor genomes" 28 : 2537-2547, 2011

39 Binder KA, "Gating of Cl− currents in protoplasts from the marine alga Valonia utricularis depends on the transmembrane Cl− gradient and is affected by enzymatic cell wall degradation" 191 : 165-178, 2003

40 Ueki S, "Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment" 4 : 71-77, 2009

41 Chang CC, "Functional analysis of two maize cDNAs encoding T7-like RNA polymerases" 11 : 911-926, 1999

42 Li JF, "FAST technique for Agrobacteriummediated transient gene expression in seedlings of Arabidopsis and other plant species" 5 : 5428-5431, 2010

43 Hong SY, "Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR" 8 : 112-122, 2008

44 De Sutter V, "Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells" 44 : 1065-1076, 2005

45 Nimchuk Z, "Eukaryotic fatty acylation drives plasma membranetargeting and enhances function of several type III effectorproteins from Pseudomonas syringae" 101 : 353-363, 2000

46 Huang CN, "Estimating viability of plant protoplasts using double and single staining" 135 : 80-87, 1986

47 Mitsuhara I, "Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants" 37 : 49-59, 1996

48 Locatelli F, "Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount" 21 : 865-871, 2003

49 Abel S, "Early genes and auxin action" 111 : 9-17, 1996

50 Hrazdina G, "Distribution of secondary plant metabolites and their biosynthetic enzymes in pea (Pisum sativum L.) leaves: anthocyanins and flavonol glycosides" 70 : 745-748, 1982

51 Subramaniam R, "Direct visualization of protein interactions in plant cells" 19 : 769-772, 2001

52 Worley CK, "Degradation of Aux/IAA proteins is essential for normal auxin signalling" 21 : 553-562, 2000

53 Huo N, "Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics" 49 : 1099-1108, 2006

54 Unver T, "Conserved microRNAs and their targets in model grass species Brachypodium distachyon" 230 : 659-669, 2009

55 Sheen J, "C4 gene expression" 50 : 187-217, 1999

56 Opanowicz M, "Brachypodium distachyon: making hay with a wild grass" 13 : 172-177, 2008

57 Draper J, "Brachypodium distachyon. A new model system for functional genomics in grasses" 127 : 1539-1555, 2001

58 Vogel J, "Brachypodium distachyon, a new model for the Triticeae, In Genetics and Genomics of the Triticeae" Springer 427-449, 2009

59 Brkljacic J, "Brachypodium as a model for the grasses: today and the future" 157 : 3-13, 2011

60 Swanson SJ, "Barley aleurone cells contain two types of vacuoles. Characterization of lytic organelles by use of fluorescent probes" 10 : 685-698, 1998

61 Yoo SD, "Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis" 2 : 1565-1572, 2007

62 Vogel JP, "Agrobacteriummediated transformation and inbred line development in the model grass Brachypodium distachyon" 85 : 199-211, 2006

63 Vain P, "Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis" 6 : 236-245, 2008

64 Ulmasov T, "Activation and repression of transcription by auxin-response factors" 96 : 5844-5849, 1999

65 Murashige T, "A revised medium for rapid growth and bio-assays with tobacco tissue culture" 15 : 473-497, 1962

66 Bradford MM, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding" 72 : 248-254, 1976

67 Hayashimoto A, "A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants" 93 : 857-863, 1990

68 Bart R, "A novel system for gene silencing using siRNAs in rice leaf and stemderived protoplasts" 2 : 13-22, 2006

69 Jin JB, "A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis" AMER SOC PLANT BIOLOGISTS 13 : 1511-1525, 2001

70 Frohnmeyer H, "A light responsive in vitro transcription system from evacuolated parsley protoplasts" 5 : 437-449, 1994

71 Chen S, "A highly efficient transient protoplast system for analyzingdefence gene expression and protein-protein interactions in rice" 7 : 417-427, 2006

72 Zhang Y, "A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes" 7 : 30-44, 2011