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Plant Physiol, December 2001, Vol. 127, pp. 1466-1475
UPDATE ON SIGNAL TRANSDUCTION
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ABSTRACT |
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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 |
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The availability of mutants,
transgenic plants, global gene expression profiles, and genomic
sequences has offered invaluable opportunities in understanding
organismal plant biology at the cellular and molecular level (Gai et
al., 2000
; Genome, 2000
; Parinov and Sundaresan, 2000
; Richmond and
Somerville, 2000
; Sussman et al., 2000
; Walbot, 2000
; Zhu and Wang,
2000
). Notably, molecular and genetic studies have discovered central
components from receptors to transcription factors in diverse plant
signal transduction pathways (Bleecker and Kende, 2000
; Gray and
Estelle, 2000
; McCarty and Chory, 2000
; Urao et al., 2000
; Dangl and
Jones, 2001
; Inoue et al., 2001
; Schroeder et al., 2001
; Tena et al.,
2001
; Zhu, 2001
). Still, many missing links exist in the plant
transduction pathways from signals to target genes.
Analogous to the mammalian tissue culture lines and transient gene
expression assays that are indispensable for the rapid progress in
discoveries of signal transduction pathways in multicellular organisms,
protoplast transient expression systems using parsley (Petroselinum crispum), maize (Zea mays), carrot
(Daucus carota), alfalfa (Medicago sativa),
Arabidopsis, and tobacco (Nicotiana tabacum) suspension
culture cells have been established. These plant cell lines offer new
opportunities to dissect signal transduction pathways involved in UV
(Lipphardt et al., 1988
), abscisic acid (ABA; Vasil et al., 1989
),
metabolite (Loake et al., 1991
), ribosomal RNA (Doelling and Pikaard,
1993
), light (Frohnmeyer et al., 1994
; Harter et al., 1994
), auxin (Liu
et al., 1994
), defense (Nurnberger et al., 1994
), and cell cycle
regulation (Evans and Bravo, 1983
; Nagata et al., 1992
; Ito et al.,
2001
). 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 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 (Kim et
al., 1993
), auxin (Abel and Theologis, 1996
), gibberellin (GA; Gubler
et al., 1999
), light, sugar, stress, auxin, hydrogen peroxide (Sheen,
1999
; Tena et al., 2001
), membrane transport (Maathuis et al., 1997
;
Bauer et al., 2000
; Hamilton et al., 2000
; Schroeder et al., 2001
), ABA
(Uno et al., 2000
), cytokinin (Hwang and Sheen, 2001
), and cell death
(Asai et al., 2000
; Bethke and Jones, 2001
) controls. With advances
made in novel protoplast assays, taking full advantage of the completed
plant genome sequences for functional genomic and proteomic analyses of
individual plant genes and their products will become a reality.
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A BRIEF HISTORY OF PROTOPLAST TRANSIENT EXPRESSION SYSTEMS |
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Forty years ago, Cocking published the first paper describing a
method for the isolation of plant protoplasts (Cocking, 1960
). A decade
later, the first successful experiment for the introduction of nucleic
acid into protoplasts was accomplished by Aoki and Takebe using tobacco
mesophyll protoplasts and tobacco mosaic virus RNA (Aoki and Takebe,
1969
). Although efficient methods for DNA transfection were not yet
available, protoplasts were already a useful tool for investigating
cell wall regeneration, cell division, embryogenesis, and
differentiation (Kao et al., 1970
; Nagata and Takebe, 1970
; Takebe,
1971
; Vasil and Vasil, 1972
), as well as for plant virus research
(Zaitlin and Beachy, 1974
). Subsequently, protoplasts were isolated
from diverse tissues and plants, and shown to retain physiological
activities and regulation. For example, freshly isolated mesophyll
protoplasts perform active photosynthesis and respiration (Edwards et
al., 1970
; Kanai and Edwards, 1973
; Podibelkowska et al., 1975
). In
barley (Hordeum vulgare) aleurone protoplasts, the
endogenous
-amylase gene is regulated by ABA and GA in parallel to
what is observed in seeds (Jacobsen and Beach, 1985
). In
broadbean (Vicia faba) guard cell protoplasts,
H+ ATPase is activated by blue light (Assmann et
al., 1985
). Protoplasts also retain cell membrane potentials similar to
intact cells and have served as a model system to study membrane
transporters. In particular, patch clamping of protoplasts is routinely
used to study ion channels and their regulation by light, stress, or hormones (Moran et al., 1984
; Schroeder et al., 1984
, 2001
; Maathuis and Sanders, 1994
; Cho and Spalding, 1996
; Bauer et al., 2000
; Downey
et al., 2000
; Hamilton et al., 2000
). Furthermore, protoplasts are
frequently used to analyze calcium signals and regulation in plant
cells (Gilroy and Jones, 1992
; Trewavas, 1999
; Pauly et al.,
2000
).
The development and improvement of protoplast transformation methods
with plasmid DNA by polyethylene glycol (PEG; Krens et al., 1982
;
Potrykus et al., 1985
; Negrutiu et al., 1987
), electroporation (Fromm
et al., 1985
; Nishiguchi et al., 1986
; Ou-Lee et al., 1986
; Hauptmann
et al., 1987
; Jones et al., 1989
), and microinjection (Hillmer et al.,
1992
) set the foundation to use protoplasts to study gene regulation
and signal transduction in plant cells. The establishment of new, and
more economical, convenient and sensitive reporter gene assays for
-glucuronidase (GUS; Jefferson et al., 1987
), chloramphenicol
acyltransferase (Seed and Sheen, 1988
), LUC (firefly luciferase;
Luehrsen et al., 1992
) and later green fluorescent protein (GFP; Sheen
et al., 1995
; Chiu et al., 1996
) for plant cells has also facilitated
the application of protoplast transient expression systems.
Following the establishment of the basic technologies, the first
demonstrations that plasmid DNA constructs carrying chimeric reporter
genes can be regulated by specific signals in transiently transformed
protoplasts were reported. These early examples include the
ABA-responsive Em promoter in rice (Oryza sativa)
protoplasts (Marcotte Jr. et al., 1988
), the UV-inducible chalcone
synthase (CHS) promoter and elicitor-responsive
pathogenesis-related (PR2) promoter in parsley protoplasts
(Lipphardt et al., 1988
; van de Locht et al., 1990
), the GA-regulated
-amylase gene promoter in oat (Avena sativa) and barley
aleurone protoplasts (Huttley and Baulcombe, 1989
; Gopalakrishnan et
al., 1991
; Jacobsen and Close, 1991
), the p-coumaric
acid-activated CHS promoter in alfalfa protoplasts (Loake et
al., 1991
), several tissue-specific promoters in tobacco and maize
protoplasts (Harkins et al., 1990
; Schaeffner and Sheen, 1991
, 1992
;
Sheen, 1991
), the feedback control of the shrunken
(Sh) promoter in maize protoplasts (Mass et al., 1990
), and
light and metabolic regulation of seven photosynthetic gene promoters
in maize mesophyll protoplasts (Sheen, 1990
).
In the past decade, a few laboratories have employed protoplast
transient expression to dissect the functions of cis-elements and
trans-factors in many essential processes and signaling pathways. These
significant studies have unraveled the control mechanisms of RNA
transcription, splicing, transport, and translation in maize and
tobacco protoplasts (Callis et al., 1987
; Gallie et al., 1987
, 1989
;
Goodall and Filipowicz, 1989
; Waibel and Filipowicz, 1990
; Doelling and
Pikaard, 1995
; Gallie and Bailey-Serres, 1997
); the involvement of VP1,
MYB, and bZIP factors in ABA and GA signaling in maize, barley,
and Arabidopsis protoplasts (McCarty et al., 1991
; Hattori et al.,
1992
; Kao et al., 1996
; Urao et al., 1996
; Gubler et al., 1999
; Uno et
al., 2000
); the function of promoters and AUX/IAA
proteins and auxin-response factors in auxin signaling in tobacco, pea
(Pisum sativum), carrot, and Arabidopsis protoplasts (Ballas
et al., 1993
; Abel and Theologis, 1994
, 1996
; Ulmasov et al., 1997a
,
1999
; Guilfoyle et al., 1998
), the elicitor- and WRKY factor-mediated
transcription regulation in parsley protoplasts (Eulgem et al., 1999
),
and the important cis-elements and transcription factors for light,
phosphate, sugar, and cell cycle regulation in maize, parsley, and
tobacco protoplasts (Sheen, 1990
, 1993
; Frohnmeyer et al., 1994
; Graham
et al., 1994
; Sadka et al., 1994
; Ni et al., 1996
; Yanagisawa and
Sheen, 1998
; Ito et al., 2001
). Recently, tobacco, maize, potato
(Solanum tuberosum), and Arabidopsis protoplast
transient expression assays have also been used to study protein
stability control (Worley et al., 2000
), retrotransposon regulation
(Pouteau et al., 1991
; Takeda et al., 1999
), protein targeting and
trafficking (Chang et al., 1999
; Kleiner et al., 1999
; Nimchuk et al.,
2000
; Jin et al., 2001
; Ueda et al., 2001
), cell death (Asai et al.,
2000
), virus movement proteins (Heinlein et al., 1995
; McLean et al.,
1995
; Huang et al., 2000
), resistance gene product (Leister and
Katagiri, 2000
), heat shock proteins and factors (Czarnecka-Verner et
al., 2000
; Kirschner et al., 2000
), protein-protein interactions
(Subramaniam et al., 2001
), and stress and hormone signaling (Sheen,
1996
, 1998
; Kovtun et al., 1998
, 2000
; Hwang and Sheen, 2001
; Tena et
al., 2001
). It is anticipated that more protoplast assays will be
developed to dissect a variety of plant signal transduction pathways.
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ADVANTAGES AND LIMITATIONS OF MESOPHYLL PROTOPLAST TRANSIENT EXPRESSION SYSTEMS |
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Much can be learned about gene function and regulation in
transient assays. Isolated mesophyll protoplasts usually represent active and homogeneous cell populations (Fig.
1) that are amenable for synchronous
pharmacological and biochemical treatments, the analysis of early and
transient responses, and most importantly for DNA transformation.
Recent extensive effort in the generation of Arabidopsis knockouts has
revealed that the majority of single gene mutants lack overt phenotypes
(Bouche and Bouchez, 2001
), suggesting the functional redundancy of
plant genes under common growth conditions. Protoplast transient
expression assays can be used for high-throughput screening of
candidate genes even for closely related members of gene families
(Sheen, 1996
, 1998
; Kovtun et al., 2000
; Cheng et al., 2001
; Tena et
al., 2001
). Constitutively active and dominant-negative mutants can be
rationally created and tested (Tena et al., 2001
). The use of epitope
and GFP tags enables gene products to be more easily followed and
studied in transiently transformed plant cells (Fig.
2; Chiu et al., 1996
; Sheen, 1996
).
Although generating transgenic plants is no longer a formidable task
for Arabidopsis (Clough and Bent, 1998
), the unpredictable nature of
transgene expression and phenotypes still requires major effort, thus
limiting the number of genes and constructs that can be analyzed
simultaneously. The transient nature of the protoplast assay can also
circumvent the difficulty in analyzing genes that cause lethality when
deleted or overexpressed in plants. In combination with the
increasingly available information on global gene expression patterns
(Richmond and Somerville, 2000
; Zhu and Wang, 2000
), transient assay
results can facilitate design of more precise and productive
experiments using transgenic and mutant plants.
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Because many plant signal transduction pathways are active in mesophyll
cells, conserved aspects of plant signaling mechanisms can be
established using these cells. The signal transduction pathways found
in mesophyll cells can potentially be generalized to other cell types,
e.g. root and meristem cells, with the addition of cell type-specific
components and/or the use of genes with homologous functions but
distinct expression patterns (Hwang and Sheen, 2001
). Recent studies
have shown that a conserved two-component cytokinin signaling pathway
established in mesophyll protoplasts is also active in the root and in
shoot meristematic cells (Hwang and Sheen, 2001
; Inoue et al., 2001
).
Mesophyll protoplasts isolated from fresh leaves have many practical
advantages. For example, plant materials are grown from seeds that are
genetically stable and more easily stored without subculturing and
without needing a sterile tissue culture facility. Non-sterile and
differentiated cells are abundant and accessible. Fast and simple
procedures have been established to obtain homogeneous, active, and
responsive mesophyll protoplasts (Fig. 1) with high transformation
efficiency (Fig. 2). The transformation efficiency of Arabidopsis and
maize mesophyll protoplasts can reach 90% (Fig. 2) and 75%,
respectively (J. Sheen, unpublished data), and cotransfection of
multiple plasmids expressing different constructs is very efficient (Abel and Theologis, 1994
; Kovtun et al., 1998
; Sheen, 1998
). Compared
with biolistic transient assays that are less effective, this high
level of transformation efficiency enables broader functional analyses
of protein products of transgenes in protoplast transient assays.
Mesophyll protoplasts can also be isolated from maize and Arabidopsis
mutants for cellular and biochemical analysis in transient assays (L. Zhou and J. Sheen, unpublished data; Asai et al., 2000
; Uno et
al., 2000
).
Despite many advantages, conceivable limitations of protoplast
transient expression systems also exist. First of all, it is presently
not possible to isolate active protoplasts from each plant cell type or
from all growth conditions (Power and Chapman, 1985
). For example,
etiolated true leaves grown in the dark can be obtained from wild-type
monocot plants such as maize and barley, but not commonly from dicot
plants such as Arabidopsis and tobacco. Currently, etiolated or
greening maize leaves provide the best source of mesophyll protoplasts
to study synchronous light and sugar regulation of photosynthetic genes
(Sheen, 1990
, 1991
, 1993
; Schaeffner and Sheen, 1991
, 1992
; Jang and
Sheen, 1994
; Yanagisawa and Sheen, 1998
). Establishment of new
physiological assays is empirical and can be time-consuming. In the
case of transgene overexpression, interpretation of the results must be
cautious. Cell walls, plasmodesmata, and cell-cell interactions are
lost or interrupted. However, with an optimal supply of nutrients and hormones, Arabidopsis mesophyll protoplasts can actually be used as
"stem cells" and the starting point to study cell wall
regeneration, cell proliferation, cell-cell communication,
embryogenesis, and differentiation (Damm et al., 1989
; Masson and
Paszkowski, 1992
; Wenck and Marton, 1995
; Luo and Koop, 1997
; Mordhorst
et al., 1998
).
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MISCONCEPTIONS |
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Although protoplast transient expression assays appear to be
simple and straightforward, proficiency requires training, lots of
patience, creativity, determination, and a "feeling" for the organism. Because protoplast isolation requires enzymatic removal of
the cell wall, there is the mistaken impression that protoplasts are
irreversibly wounded and thus are stressed and dying cells. When
properly isolated and maintained, protoplasts retain their original
biochemical and cellular activities. Based on vital staining with
fluorescein diacetate (Larkin, 1976
) or Evans blue (Asai et al., 2000
),
the viability of freshly isolated intact maize and Arabidopsis
mesophyll protoplasts is greater than 95% (Fig. 1) for more than
48 h in simple mannitol solution. Most transient assays can be
carried out within 12 h after isolation. In fact, Arabidopsis
mesophyll protoplasts were used recently as an experimental system to
study a specific cell death program induced by fumonisin B1 toxin (Asai
et al., 2000
). Barley aleurone protoplasts are conducive to study cell
death program mediated by reactive oxygen species (Bethke and Jones,
2001
). The best evidence against the perception that protoplasts are
highly stressed and not suitable for studying signaling is the
demonstration that maize and Arabidopsis protoplasts respond to
oxidative, heat, and osmotic stress signals and pathogen-derived
elicitors as do cells in intact plants (Nurnberger et al., 1994
; Sheen,
1996
; Kovtun et al., 1998
, 2000
; Eulgem et al., 1999
; Tena et al.,
2001
). Thus, the "stress" status of mesophyll protoplasts can be
quantified using stress-inducible genes. If protoplasts are truly
stressed and dying, the general gene expression program is shut down
(Asai et al., 2000
; J. Sheen, unpublished data). Furthermore,
the functionality of plasma membrane proteins in Arabidopsis mesophyll
protoplasts has been demonstrated by the detection of cell surface
receptor activities for cytokinin and for a peptide elicitor (Hwang and
Sheen, 2001
; Tena et al., 2001
).
For successful and reproducible results, great care should be taken in
establishing plant growth conditions (Power and Chapman, 1985
; Masson
and Paszkowski, 1992
), monitoring leaf morphology, age and development,
isolating protoplasts, and in testing and comparing various
physiological responses between transgenes in protoplasts and
endogenous genes in protoplasts and intact plants. Homogeneous
populations (>95%) of mesophyll protoplasts are routinely obtained;
purity can be easily confirmed by microscopic observation (Fig. 1;
Sheen, 1995
). Other leaf cell types are generally not released using
the established procedure for mesophyll protoplast isolation (Sheen,
1995
). Other experimental conditions such as plasmid DNA purity, DNA to
protoplast ratio, and protoplast culture density need to be optimized.
The method of choice for DNA transfection needs to be tested
empirically. For instance, electroporation for maize mesophyll
protoplasts and PEG transfection for Arabidopsis mesophyll protoplasts
work well (Fig. 2; Sheen, 1990
, 1991
; Kovtun et al., 2000
; Hwang and
Sheen, 2001
). For each transfection sample, 100 times fewer protoplasts
than previously established (106-7) gives
optimal gene expression based on the activity of constitutive 35S and
ubiquitin promoters (J. Sheen, unpublished data). Depending on the nature of transient expression analysis, one million mesophyll protoplasts could be used for 100 or more transfections and/or assays,
a substantial efficiency improvement if plant material is limited. The
activities of single cells can also be easily monitored and visualized
by vital markers, such as GFP and LUC (Chiu et al., 1996
; Sheen, 1996
;
Kovtun et al., 1998
; Yanagisawa and Sheen, 1998
; Hwang and Sheen, 2001
;
Zhu, 2001
). Although responses in transient expression assays can be
monitored as early as 1 to 2 h after DNA transfection, optimal
assay conditions need to be established experimentally. In general,
protoplast transient expression analysis is intensive and demanding but
enormously rewarding.
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DISCOVER AND DISSECT PLANT SIGNAL TRANSDUCTION PATHWAYS |
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The best demonstration of the fitness of the protoplast transient
expression systems for discovering and dissecting plant signal
transduction pathways is to provide successful examples (Fig.
3). These studies support the idea that
key regulators of plant signaling transduction pathways are conserved
in dicots and monocots, and justify the use of model plants such as
maize and Arabidopsis. For instance, the discovery of the global sugar repression of photosynthetic gene promoters in maize mesophyll protoplasts is now supported by studies in diverse plant species (Sheen, 1990
; Sheen et al., 1999
). The proposed role of hexokinase as a
sugar sensor based on maize protoplast transient expression analysis is
also validated by transgenic plant studies (Jang et al., 1997
; Dai et
al., 1999
) and by the isolation of hexokinase mutants displaying Glc
insensitivity in Arabidopsis (Sheen et al., 1999
). The negative role of
ABA insensitive protein (ABI1) and the redundancy of protein
phosphatase 2C (PP2Cs) in ABA signaling revealed by transient
expression analysis in maize mesophyll protoplasts (Sheen, 1998
) have
been confirmed by the isolation of Arabidopsis abi1 null mutants (Gosti
et al., 1999
) and by studies in a rice protoplast assay (Hagenbeek et
al., 2000
).
|
Protoplast transient expression analysis has been used for intensive
studies of auxin regulated gene expression in diverse plants (Ballas et
al., 1993
; Abel and Theologis, 1996
; Guilfoyle et al., 1998
; Ulmasov et
al., 1999
). A soybean (Glycine max) GH3 promoter
exhibits similar auxin induction in tobacco, carrot, maize and
Arabidopsis protoplasts, suggesting conservation in auxin signaling
(Liu et al., 1994
; Guilfoyle et al., 1998
; Kovtun et al., 1998
, 2000
).
Recent characterization of Arabidopsis auxin signaling mutants
(Gray and Estelle, 2000
) supports the physiological functions of
AUX/IAA proteins and auxin-response factors first discovered in
protoplast transient expression assays (Abel and Theologis, 1996
;
Ulmasov et al., 1997a
, 1997b
; Guilfoyle et al., 1998
; Ulmasov et al.,
1999
). The alternation of IAA17/AXR3 mutant protein stability has also
been demonstrated using a protoplast transient assay (Worley et al.,
2000
). Mesophyll protoplasts have been isolated from Arabidopsis
mutants (jar1, etr1, pad4,
npr1, acd2, cpr1, and cpr6)
and transgenic plants (NahG) to investigate fumonisin
B1-induced cell death program that requires ethylene, salicylate, and
jasmonate signaling pathways (Asai et al., 2000
). The activity of an
ABA-regulated reporter gene in a protoplast transient expression assay
has been shown to be repressed in the ABA insensitive mutants,
abi1 and abi2, but greatly enhanced in the ABA
hypersensitive mutant era1 (Uno et al., 2000
). Recently, the
use of the maize and Arabidopsis mesophyll protoplast transient expression assays has allowed functional analysis of the MAPK signaling
cascades involved in oxidative stress, auxin, and defense signaling
pathways (Kovtun et al., 1998
; Kovtun et al., 2000
; Tena et al., 2001
).
Finally, we have established a quantitative and specific protoplast
assay based on cytokinin early response gene transcription (D'Agostino
et al., 2000
; Hwang and Sheen, 2001
). Using this novel system, we have
identified a two-component circuitry in Arabidopsis cytokinin signal
transduction consisting of four major steps: His protein kinase
receptor sensing and signaling, phosphotransmitter nuclear
translocation, response regulator-dependent transcription activation,
and a negative feedback loop through cytokinin-inducible genes encoding
a distinct class of response regulators (Hwang and Sheen, 2001
).
Analyses of transgenic tissues and plants support the importance of
this central signaling pathway in diverse cytokinin responses. This
protoplast-based analysis is consistent with genetic characterization
of Arabidopsis cytokinin mutants cki1 and cre1
(Kakimoto, 1996
; Hwang and Sheen, 2001
; Inoue et al., 2001
) and with
cytokinin-inducible gene regulation in wild-type and transgenic plants
(D'Agostino et al., 2000
).
The development of various protoplast transient expression assays has broadened the methodology for plant signaling pathway analyses to include biochemical, cellular, genomics, genetic, and transgenic tools. In most cases, discoveries made in protoplasts and conclusions derived from transient expression assays have been supported by transgenic plant studies and/or the isolation and characterization of relevant mutants. It will be possible to use protoplasts isolated from mutants for gene cloning by functional complementation with appropriate transient assays.
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FUTURE PERSPECTIVES |
|---|
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|---|
Powerful and versatile cell systems using mesophyll protoplasts
isolated from fresh leaves of maize and Arabidopsis have been developed. These protoplast transient expression systems show regulated
gene expression in response to internal and external signals and allow
efficient and penetrating analysis of molecular mechanisms underlying
hormone, sugar, stress, and defense signaling (Fig. 3). The use of a
combination of tools and diverse resources in the protoplast system
offers unprecedented opportunities to answer questions in plant
physiology and development. Similar protoplast systems could also be
developed using tobacco, barley, wheat (Triticum
aestivum), and rice mesophyll protoplasts (J. Sheen,
unpublished). The applications of protoplast transient expression
systems will continue to contribute to the elucidation of intracellular
signaling mechanisms in plants. Observation, imagination, creativity,
and commitment are necessary for making discoveries using the
protoplast assays. In the model plant Arabidopsis, extensive genetic
analyses, genomic sequences, and global gene expression profiles offer
a wealth of information to test signal transduction mechanisms in the
protoplast transient expression assays (Fig. 3). The conclusions
derived from single cell studies can then be readily confirmed using
transgenic plants and mutants. It is now possible to develop
high-throughout protoplast transient assays for functional genomic and
proteomic research, such as to screen for activities and functions of
protein kinases, protein phosphatases, receptors, G proteins, and
transcription factors (Chory and Wu, 2001
; Hwang and Sheen, 2001
; Tena
et al., 2001
), as well as protein kinase substrates (Cheng et al.,
2001
). Studies in protoplast systems can provide a framework for whole
plant analysis of tissue- or cell type-specific pathways in knockout mutants and transgenic plants.
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ACKNOWLEDGMENTS |
|---|
I apologize for overlooking work on plant protoplasts not cited in this review. I would like to thank Brandon Moore, Heven Sze, Virginia Walbot, and Natasha V. Raikhel for valuable discussion and comments on the review, and my past and present colleagues, Anton Schaeffner, Hai Huang, Jyun-Chyun Jang, Patricia Leon, Kin-Ing To, Wan-Ling Chiu, Weike Zeng, Helen Wang, Yelena Kovtun, Shu-Hua Cheng, Brandon Moore, Guillaume Tena, Tsuneaki Asai, Ildoo Hwang, Matthew Willmann, Filip Rolland, and Senthil Ramu, who took the adventure with me in testing and improving the maize and Arabidopsis mesophyll protoplast systems. The reporters 35S-CAT/LUC, AtUBQ10-GUS, ZmUBQ-GUS, and Actin-GUS were kindly provided by Virginia Walbot, Judy Callis, Peter Quail, and Ray Wu, respectively.
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FOOTNOTES |
|---|
Received September 7, 2001; accepted September 24, 2001.
1 This work was supported by the National Science Foundation, by the U.S. Department of Agriculture, by the National Institutes of Health, and by Hoechst A.G.
* E-mail sheen{at}molbio.mgh.harvard.edu; fax 617-726-6893.
www.plantphysiol.org/cgi/doi/10.1104/pp.010820.
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LITERATURE CITED |
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-Amy-2 promoter is regulated by gibberellin in transformed oat aleurone protoplasts.
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Nature
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leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo.
Plant J
22: 345-354[CrossRef][Web of Science][Medline]This article has been cited by other articles:
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M. C. Pomeranz, C. Hah, P.-C. Lin, S. G. Kang, J. J. Finer, P. J. Blackshear, and J.-C. Jang The Arabidopsis Tandem Zinc Finger Protein AtTZF1 Traffics between the Nucleus and Cytoplasmic Foci and Binds Both DNA and RNA Plant Physiology, January 1, 2010; 152(1): 151 - 165. [Abstract] [Full Text] [PDF] |
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R. L. McCarthy, R. Zhong, and Z.-H. Ye MYB83 Is a Direct Target of SND1 and Acts Redundantly with MYB46 in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis Plant Cell Physiol., November 1, 2009; 50(11): 1950 - 1964. [Abstract] [Full Text] [PDF] |
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L. Moeller, Q. Gan, and K. Wang A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway J. Exp. Bot., August 1, 2009; 60(12): 3337 - 3352. [Abstract] [Full Text] [PDF] |
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Y. T. Cheng, H. Germain, M. Wiermer, D. Bi, F. Xu, A. V. Garcia, L. Wirthmueller, C. Despres, J. E. Parker, Y. Zhang, et al. Nuclear Pore Complex Component MOS7/Nup88 Is Required for Innate Immunity and Nuclear Accumulation of Defense Regulators in Arabidopsis PLANT CELL, August 1, 2009; 21(8): 2503 - 2516. [Abstract] [Full Text] [PDF] |
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S. Xia, Z. Zhu, L. Hao, J.-G. Chen, L. Xiao, Y. Zhang, and X. Li Negative Regulation of Systemic Acquired Resistance by Replication Factor C Subunit3 in Arabidopsis Plant Physiology, August 1, 2009; 150(4): 2009 - 2017. [Abstract] [Full Text] [PDF] |
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C.-W. Cheng, Y.-Y. Hsiao, H.-C. Wu, C.-M. Chuang, J.-S. Chen, C.-H. Tsai, Y.-H. Hsu, Y.-C. Wu, C.-C. Lee, and M. Meng Suppression of Bamboo Mosaic Virus Accumulation by a Putative Methyltransferase in Nicotiana benthamiana J. Virol., June 1, 2009; 83(11): 5796 - 5805. [Abstract] [Full Text] [PDF] |
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H. Lin, Y. Yang, R. Quan, I. Mendoza, Y. Wu, W. Du, S. Zhao, K. S. Schumaker, J. M. Pardo, and Y. Guo Phosphorylation of SOS3-LIKE CALCIUM BINDING PROTEIN8 by SOS2 Protein Kinase Stabilizes Their Protein Complex and Regulates Salt Tolerance in Arabidopsis PLANT CELL, May 1, 2009; 21(5): 1607 - 1619. [Abstract] [Full Text] [PDF] |
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B. O.R. Bargmann and K. D. Birnbaum Positive Fluorescent Selection Permits Precise, Rapid, and In-Depth Overexpression Analysis in Plant Protoplasts Plant Physiology, March 1, 2009; 149(3): 1231 - 1239. [Abstract] [Full Text] [PDF] |
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J.-I. Cho, N. Ryoo, J.-S. Eom, D.-W. Lee, H.-B. Kim, S.-W. Jeong, Y.-H. Lee, Y.-K. Kwon, M.-H. Cho, S. H. Bhoo, et al. Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors Plant Physiology, February 1, 2009; 149(2): 745 - 759. [Abstract] [Full Text] [PDF] |
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J. Zhou, C. Lee, R. Zhong, and Z.-H. Ye MYB58 and MYB63 Are Transcriptional Activators of the Lignin Biosynthetic Pathway during Secondary Cell Wall Formation in Arabidopsis PLANT CELL, January 1, 2009; 21(1): 248 - 266. [Abstract] [Full Text] [PDF] |
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A. Almagro, S. H. Lin, and Y. F. Tsay Characterization of the Arabidopsis Nitrate Transporter NRT1.6 Reveals a Role of Nitrate in Early Embryo Development PLANT CELL, December 1, 2008; 20(12): 3289 - 3299. [Abstract] [Full Text] [PDF] |
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F. J. Sandoval, Y. Zhang, and S. Roje Flavin Nucleotide Metabolism in Plants: MONOFUNCTIONAL ENZYMES SYNTHESIZE FAD IN PLASTIDS J. Biol. Chem., November 7, 2008; 283(45): 30890 - 30900. [Abstract] [Full Text] [PDF] |
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R. Zhong, C. Lee, J. Zhou, R. L. McCarthy, and Z.-H. Ye A Battery of Transcription Factors Involved in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis PLANT CELL, October 1, 2008; 20(10): 2763 - 2782. [Abstract] [Full Text] [PDF] |
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S.-H. Lin, H.-F. Kuo, G. Canivenc, C.-S. Lin, M. Lepetit, P.-K. Hsu, P. Tillard, H.-L. Lin, Y.-Y. Wang, C.-B. Tsai, et al. Mutation of the Arabidopsis NRT1.5 Nitrate Transporter Causes Defective Root-to-Shoot Nitrate Transport PLANT CELL, September 1, 2008; 20(9): 2514 - 2528. [Abstract] [Full Text] [PDF] |
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X. Li, S. Chanroj, Z. Wu, S. M. Romanowsky, J. F. Harper, and H. Sze A Distinct Endosomal Ca2+/Mn2+ Pump Affects Root Growth through the Secretory Process Plant Physiology, August 1, 2008; 147(4): 1675 - 1689. [Abstract] [Full Text] [PDF] |
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C. Denoux, R. Galletti, N. Mammarella, S. Gopalan, D. Werck, G. De Lorenzo, S. Ferrari, F. M. Ausubel, and J. Dewdney Activation of Defense Response Pathways by OGs and Flg22 Elicitors in Arabidopsis Seedlings Mol Plant, May 22, 2008; (2008) ssn019v1. [Abstract] [Full Text] [PDF] |
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D. Panikashvili, S. Savaldi-Goldstein, T. Mandel, T. Yifhar, R. B. Franke, R. Hofer, L. Schreiber, J. Chory, and A. Aharoni The Arabidopsis DESPERADO/AtWBC11 Transporter Is Required for Cutin and Wax Secretion Plant Physiology, December 1, 2007; 145(4): 1345 - 1360. [Abstract] [Full Text] [PDF] |
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J. Zhao, W. Zhang, Y. Zhao, X. Gong, L. Guo, G. Zhu, X. Wang, Z. Gong, K. S. Schumaker, and Y. Guo SAD2, an Importin -Like Protein, Is Required for UV-B Response in Arabidopsis by Mediating MYB4 Nuclear Trafficking PLANT CELL, November 1, 2007; 19(11): 3805 - 3818. [Abstract] [Full Text] [PDF] |
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J. M. Hernandez, A. Feller, K. Morohashi, K. Frame, and E. Grotewold The basic helix loop helix domain of maize R links transcriptional regulation and histone modifications by recruitment of an EMSY-related factor PNAS, October 23, 2007; 104(43): 17222 - 17227. [Abstract] [Full Text] [PDF] |
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J. Aker, R. Hesselink, R. Engel, R. Karlova, J. W. Borst, A. J.W.G. Visser, and S. C. de Vries In Vivo Hexamerization and Characterization of the Arabidopsis AAA ATPase CDC48A Complex Using Forster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy and Fluorescence Correlation Spectroscopy Plant Physiology, October 1, 2007; 145(2): 339 - 350. [Abstract] [Full Text] [PDF] |
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R. Zhong, E. A. Richardson, and Z.-H. Ye The MYB46 Transcription Factor Is a Direct Target of SND1 and Regulates Secondary Wall Biosynthesis in Arabidopsis PLANT CELL, September 1, 2007; 19(9): 2776 - 2792. [Abstract] [Full Text] [PDF] |
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J. Rosler, I. Klein, and M. Zeidler Arabidopsis fhl/fhy1 double mutant reveals a distinct cytoplasmic action of phytochrome A PNAS, June 19, 2007; 104(25): 10737 - 10742. [Abstract] [Full Text] [PDF] |
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A. T. Fuglsang, Y. Guo, T. A. Cuin, Q. Qiu, C. Song, K. A. Kristiansen, K. Bych, A. Schulz, S. Shabala, K. S. Schumaker, et al. Arabidopsis Protein Kinase PKS5 Inhibits the Plasma Membrane H+-ATPase by Preventing Interaction with 14-3-3 Protein PLANT CELL, May 1, 2007; 19(5): 1617 - 1634. [Abstract] [Full Text] [PDF] |
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R. Quan, H. Lin, I. Mendoza, Y. Zhang, W. Cao, Y. Yang, M. Shang, S. Chen, J. M. Pardo, and Y. Guo SCABP8/CBL10, a Putative Calcium Sensor, Interacts with the Protein Kinase SOS2 to Protect Arabidopsis Shoots from Salt Stress PLANT CELL, April 1, 2007; 19(4): 1415 - 1431. [Abstract] [Full Text] [PDF] |
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A. M. Burza, I. Pekala, J. Sikora, P. Siedlecki, P. Malagocki, M. Bucholc, L. Koper, P. Zielenkiewicz, M. Dadlez, and G. Dobrowolska Nicotiana tabacum Osmotic Stress-activated Kinase Is Regulated by Phosphorylation on Ser-154 and Ser-158 in the Kinase Activation Loop J. Biol. Chem., November 10, 2006; 281(45): 34299 - 34311. [Abstract] [Full Text] [PDF] |
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Y. Miao, D. Lv, P. Wang, X.-C. Wang, J. Chen, C. Miao, and C.-P. Song An Arabidopsis Glutathione Peroxidase Functions as Both a Redox Transducer and a Scavenger in Abscisic Acid and Drought Stress Responses PLANT CELL, October 1, 2006; 18(10): 2749 - 2766. [Abstract] [Full Text] [PDF] |
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J. B. Heo, H. S. Rho, S. W. Kim, S. M. Hwang, H. J. Kwon, M. Y. Nahm, W. Y. Bang, and J. D. Bahk OsGAP1 Functions as a Positive Regulator of OsRab11-mediated TGN to PM or Vacuole Trafficking Plant Cell Physiol., December 1, 2005; 46(12): 2005 - 2018. [Abstract] [Full Text] [PDF] |
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E. R. Havecker, X. Gao, and D. F. Voytas The Sireviruses, a Plant-Specific Lineage of the Ty1/copia Retrotransposons, Interact with a Family of Proteins Related to Dynein Light Chain 8 Plant Physiology, October 1, 2005; 139(2): 857 - 868. [Abstract] [Full Text] [PDF] |
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L.-z. Tao, A. Y. Cheung, C. Nibau, and H.-m. Wu RAC GTPases in Tobacco and Arabidopsis Mediate Auxin-Induced Formation of Proteolytically Active Nuclear Protein Bodies That Contain AUX/IAA Proteins PLANT CELL, August 1, 2005; 17(8): 2369 - 2383. [Abstract] [Full Text] [PDF] |
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M. Endo, S. Nakamura, T. Araki, N. Mochizuki, and A. Nagatani Phytochrome B in the Mesophyll Delays Flowering by Suppressing FLOWERING LOCUS T Expression in Arabidopsis Vascular Bundles PLANT CELL, July 1, 2005; 17(7): 1941 - 1952. [Abstract] [Full Text] [PDF] |
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E. Russinova, J.-W. Borst, M. Kwaaitaal, A. Cano-Delgado, Y. Yin, J. Chory, and S. C. de Vries Heterodimerization and Endocytosis of Arabidopsis Brassinosteroid Receptors BRI1 and AtSERK3 (BAK1) PLANT CELL, December 1, 2004; 16(12): 3216 - 3229. [Abstract] [Full Text] [PDF] |
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Q. Wang, R. W. Sullivan, A. Kight, R. L. Henry, J. Huang, A. M. Jones, and K. L. Korth Deletion of the Chloroplast-Localized Thylakoid Formation1 Gene Product in Arabidopsis Leads to Deficient Thylakoid Formation and Variegated Leaves Plant Physiology, November 1, 2004; 136(3): 3594 - 3604. [Abstract] [Full Text] [PDF] |
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M.-H. Han, S. Goud, L. Song, and N. Fedoroff The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation PNAS, January 27, 2004; 101(4): 1093 - 1098. [Abstract] [Full Text] [PDF] |
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K. Birnbaum, D. E. Shasha, J. Y. Wang, J. W. Jung, G. M. Lambert, D. W. Galbraith, and P. N. Benfey A Gene Expression Map of the Arabidopsis Root Science, December 12, 2003; 302(5652): 1956 - 1960. [Abstract] [Full Text] [PDF] |
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W. Zhang, C. Wang, C. Qin, T. Wood, G. Olafsdottir, R. Welti, and X. Wang The Oleate-Stimulated Phospholipase D, PLD{delta}, and Phosphatidic Acid Decrease H2O2-Induced Cell Death in Arabidopsis PLANT CELL, October 1, 2003; 15(10): 2285 - 2295. [Abstract] [Full Text] [PDF] |
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K. H. Lee, S. J. Kim, Y. J. Lee, J. B. Jin, and I. Hwang The M Domain of atToc159 Plays an Essential Role in the Import of Proteins into Chloroplasts and Chloroplast Biogenesis J. Biol. Chem., September 19, 2003; 278(38): 36794 - 36805. [Abstract] [Full Text] [PDF] |
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E. J. Sohn, E. S. Kim, M. Zhao, S. J. Kim, H. Kim, Y.-W. Kim, Y. J. Lee, S. Hillmer, U. Sohn, L. Jiang, et al. Rha1, an Arabidopsis Rab5 Homolog, Plays a Critical Role in the Vacuolar Trafficking of Soluble Cargo Proteins PLANT CELL, May 1, 2003; 15(5): 1057 - 1070. [Abstract] [Full Text] |
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F. M. Ausubel Summaries of National Science Foundation-Sponsored Arabidopsis 2010 Projects and National Science Foundation-Sponsored Plant Genome Projects That Are Generating Arabidopsis Resources for the Community Plant Physiology, June 1, 2002; 129(2): 394 - 437. [Full Text] [PDF] |
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F. Rolland, B. Moore, and J. Sheen Sugar Sensing and Signaling in Plants PLANT CELL, May 1, 2002; 14(90001): S185 - 205. [Full Text] [PDF] |
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