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Signal Transduction in Arabidopsis and Maize Mesophyll Protoplasts
Plant protoplasts show physiological
perceptions and responses to hormones, metabolites, environmental
cues, and pathogen-derived elicitors, similar to cell-autonomous
responses in intact tissues and plants. The development of defined
protoplast transient expression systems for high throughput screening
and systematic characterization of gene functions has greatly contributed
to elucidating plant signal transduction pathways, in combination
with genetic, genomic, and transgenic approaches. |
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INTRODUCTION
The availability of mutants, transgenic plants, global
gene expression profiles, and genomic sequences has offered invaluable
opportunities for understanding organismal plant biology at the
cellular and molecular level. Notably, molecular and genetic studies
have discovered central components from receptors to transcription
factors in diverse plant signal transduction pathways. Still, many
missing links exist in the plant transduction pathways. |
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Tissue culture cell lines and transient expression
assays have proven indispensable for rapid progress in signal transduction
research in mammals. Analogous systems in plants have been established
that use protoplast transient expression of parsley, maize, carrot,
alfalfa, Arabidopsis, and tobacco suspension culture cells. These
plant cell lines offer new opportunities to dissect signal transduction
pathways involved in UV, ABA (abscisic acid), metabolite, ribosomal
RNA, light, auxin, defense, and cell cycle regulation. |
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Compared with cell culture lines, the use of fresh
tissues as protoplast sources offers unique advantages. For example,
protoplasts isolated from plant tissues retain their cell identity
and differentiated state. They also show high transformation efficiency
with low maintenance. These freshly isolated protoplasts have proven
to be physiological and versatile cell systems for studying a broad
spectrum of plant signaling mechanisms underlying phytochrome, clock,
auxin, GA, light, sugar, stress, auxin, H2O2,
membrane transport, ABA, cytokinin, and cell death controls. Advances
in novel protoplast assays will help make functional genomic and
proteomic analyses of individual plant genes and their products
a reality. They also provide a convenient and powerful tool for
the cellular, genetic and molecular analysis and complementation of existing mutants. |
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We are providing two protoplast protocols
detailing the preparation and selection of plant materials, protoplast
isolation and incubation procedures, DNA transfection methods, and
reporter gene assays, as well as key references and trouble-shooting
tips. A more general introduction to the maize and Arabidopsis mesophyll
protoplast systems is provided in a recent review (Sheen, Plant
Physiol. 127: 1466-1475, 2001 PDF).
A "FAQ" website focusing on the use of mesophyll
protoplast systems has been posted. |
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The protocol has been streamlined and
can be applied to different types of plant materials. The growth
condition of plants seems to be the most critical factor for experimental
reproducibility. Each lab may need to work out the best plant growth
conditions for its setting. It usually takes some time to develop
specific assays since these protoplasts are dynamic with numerous
endogenous activities (a physiological system!). You may need to
modify plant growth conditions or define specific physiological
and developmental stages for protoplast isolation. It certainly
helps if you think from a plant's point of view!! The protocol is
simple and the approach is powerful. However, it only awards success
to the scientists who are willing to take the time to master the
system with patience and faith. Good luck with your experiments!
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References for Arabidopsis : |
He et al., Cell, 125: 563-575
(2006) (immunity) |
Rashotte et al., PNAS, 103: 11081-11085
(2006) (cytokinin) |
Ehlert et al., The Plant J, 46: 890-900
(2006) (protoplast two hybrid) |
Tiwari et al., Methods Mol Biol, 323: 237-244
(2006) (auxin) |
Kim et al., PNAS, 103: 814-819
(2006) (cytokinin) |
De Sutter et al., The Plant J, 44: 1065-1076
(2005) (JA/tobacco BY2) |
Kim et al., The Plant Cell, 17: 888-902
(2005) (actin/vacuole/Golgi) |
Tao et al., The Plant Cell, 17: 2369-2383
(2005) (auxin RAC) |
Fujita et al., The Plant Cell, 17: 3470-3488
(2005) (ABA) |
Wang et al., The Plant Cell, 17: 1979-1993
(2005) (auxin ARF7) |
Pati et al., Protoplasma, 226: 217-221
(2005) (regeneration/tobacco/Lotus) |
Boudsocq et al., JBC, 279: 41758-41766
(2004) (SnRK2, ABA/osmotic) |
Russinova et al., The Plant Cell, 16: 3216-3229
(2004) (BR) |
Sohn et al., The Plant Cell, 15: 1057-1070
(2003) (trafficking) |
Birnbaum et al., Science, 302: 1956-1960
(2003) (root map) |
Yanagisawa et al., Nature, 425: 521-525
(2003) (glucose/ethylene) |
Asai et al., Nature, 415: 977-983
(2002) (immunity) |
Lee et al., The Plant Cell, 13: 2175-2190
(2001) (targeting) |
Jin et al., The Plant Cell, 13: 1511-1525
(2001) (trafficking) |
Sheen., Plant Physiol., 127:1466-1475
(2001) (review) |
Hwang & Sheen, Nature, 413: 383-389
(2001) (cytokinin) |
Uno et al., PNAS, 19: 11632-11637
(2000) (ABA) |
Kovtun et al., PNAS, 97: 2940-2945
(2000) (H2O2, auxin) |
Worley et al., The Plant J, 21: 553-562
(2000) (IAA/Aux degradation/tobacco) |
Abel & Theologis., Plant J., 5: 421-427
(1994) (auxin) |
Masson & Paszkowski., Plant J., 2: 829-833
(1992) (regeneration) |
Schäffner & Sheen, The Plant Cell, 3: 997-1012
(1991) (light/RbcS/tobacco) |
Damm et al., MGG, 217: 6-12
(1989) (regeneration) |
Negrutiu et al., Plant Mol Biol, 8: 363-373
(1987) (tobacco/PEG method) |
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References for Maize: |
Yanagisawa et al., Nature, 425: 521-525
(2003) (glucose/ethylene) |
Moore et al., Science, 300: 332-336
(glucose) |
Sheen, Plant Physiol, 127: 1466-1475
(2001) (review) |
Cheng et al., FEBS Letters, 503: 185-188
(CDPK/PAL) |
Cheng et al., Plant Cell,11: 911-926
(1999) (chloroplast/mitochondria targeting) |
Kovtun et al., Nature, 385: 716-720
(1998) (auxin) |
Yanagisawa and Sheen., Plant Cell, 10: 75-89
(1998) (light) |
Sheen, PNAS, 98: 975-980
(1998) (ABA) |
Sheen, Science, 274: 1900-1902
(1996) (ABA/stresses) |
Chiu et al., Current Biol, 6: 325-330
(1996) (GFP) |
Sheen et al., Plant J, 8: 777-784
(1995) (GFP) |
Sheen, Methods in Plant Cell Biology Part A, Galbraith et al., (eds).
Academic Press, Orlando. pp. 305-314
(1995) (methods) |
Jang and Sheen. Plant Cell 6: 1665-1679
(1994) (glucose) |
Sheen. EMBO J. 12: 221-232
(1993) (light) |
Schäffner & Sheen, Plant J, 2: 221-232
(1992) (light/PEPC) |
Schäffner & Sheen, The Plant Cell, 3: 997-1012
(1991) (light/RBCS) |
Sheen. Plant Cell 3: 225-245 (1991) |
Sheen. Plant Cell 2: 1027-1038
(1990) |
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References for Rice: |
Kobayashi et al., Plant Cell, 16: 1163-1177
(2004) (OsSnRK, ABA/osmotic)
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