Breakthrough Technologies
Reliable Transient Transformation of Intact Maize
Leaf Cells for Functional Genomics and
Experimental Study1[W][OA]
Daniel R. Kirienko, Anding Luo, and Anne W. Sylvester*
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071
Maize (Zea mays) transformation routinely produces stable transgenic lines essential for functional genomics; however, transient
expression of target proteins in maize cells is not yet routine. Such techniques are critical for rapid testing of transgene constructs
and for experimental studies. Here, we report bombardment methods that depend on leaf developmental stage and result in
successful expression with broad applications. Fluorescent marker genes were constructed and bombarded into five
developmental regions in a growing maize leaf. Expression efficiency was highest in the basal-most 3 cm above the ligule of
an approximately 50-cm growing adult leaf. Straightforward dissection procedures provide access to the receptive leaf regions,
increasing efficiency from less than one transformant per cm2 to over 21 transformants per cm2. Successful expression was
routine for proteins from full genomic sequences driven by native regulatory regions and from complementary DNA sequences
driven by the constitutive maize polyubiquitin promoter and a heterologous terminator. Four tested fusion proteins, maize
PROTEIN DISULFIDE ISOMERASE-Yellow Fluorescent Protein, GLOSSY8a-monomeric Red Fluorescent Protein and maize
XYLOSYLTRANSFERASE, and maize Rho-of-Plants7-monomeric Teal Fluorescent Protein, localized as predicted in the
endoplasmic reticulum, Golgi, and plasma membrane, respectively. Localization patterns were similar between transient and
stable modes of expression, and cotransformation was equally successful. Coexpression was also demonstrated by transiently
transforming cells in a stable line expressing a second marker protein, thus increasing the utility of a single stable transformant.
Given the ease of dissection procedures, this method replaces heterologous expression assays with a more direct, native, and
informative system, and the techniques will be useful for localization, colocalization, and functional studies.
Many reporter systems depend on the temporary
observation of protein expression for experimental,
functional, or cell biological study. In some cases,
transient expression assays may be a necessary first
test for transgene expression before the generation of
stable transgenic lines. In maize (Zea mays) and other
plants with long generation times, transient expression
tests are particularly important because of the time and
expense involved in waiting for stable expression.
Transient assays in planta also provide novel experimental tools for physiological and localization studies.
Although routine in most dicotyledons (dicots) and in
some grasses, transient assays have been surprisingly
problematic in maize. For this reason, onion (Allium
cepa) epidermis may be used for gene expression
studies in monocotyledons (monocots; Amien et al.,
2010), but this approach is not ideal due to the
1
This work was supported by the National Science Foundation
(grant nos. 0501862 and 1027445) and the Department of Energy
(grant no. DE–FG02–05ER15655).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Anne W. Sylvester ([email protected]).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.199737
phylogenetic distance between the families. Recently,
grasses such as Setaria, Brachypodium, and other pooids
have been used as more closely related models for
maize functional genomics (Brutnell et al., 2010). There
remains a need, however, for a reliable transient expression method for DNA constructs in maize. Such an
advance would contribute to functional protein studies
for maize and allow for experiments that exploit the
fully sequenced maize genome (Gore et al., 2009;
Mohanty et al., 2009a, 2009b; Schnable et al., 2009, 2011;
Springer et al., 2009; Swanson-Wagner et al., 2009; Zhou
et al., 2009; Eveland et al., 2010; Li et al., 2010; Zheng
et al., 2010; Sekhon et al., 2011).
The earliest successes in plant transformation were
in dicots and involved stable, rather than transient,
expression of transgenes. These methods used Agrobacterium tumefaciens to deliver the desired DNA into
the plant nucleus (Wullems et al., 1981; Bevan et al.,
1983; De Block et al., 1984; Horsch et al., 1984). Later
methods, including whole-leaf infiltration of Agrobacterium, made both stable and transient transformation straightforward and led to significant advances
(Lloyd et al., 1986; Sheikholeslam and Weeks, 1987;
Bechtold et al., 1993; Yang et al., 2000; Haake et al.,
2002; Lu et al., 2002; Sheahan et al., 2004; Tsuda et al.,
2012). Monocots, in contrast, were initially resistant to
Agrobacterium infection, so particle bombardment was
used for both stable and transient transformation in
grasses (Oard et al., 1990; Songstad et al., 1995; Schenk
et al., 1998). It was found that bombardment promoted
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Kirienko et al.
multiple insertion events, and transgene silencing occurred (Klein et al., 1988; Vasil, 1994; Kumpatla et al.,
1997) until new Agrobacterium vectors were designed
for use in grasses (Hiei et al., 1994; Ishida et al., 1996;
Cheng et al., 1997; Tingay et al., 1997; Zhao et al., 2000;
Manickavasagam et al., 2004). New binary vectors and
other technical progress increased efficiency and reduced the number of transgene insertions; subsequently, Agrobacterium-based transformation became
routine in maize, although success was limited to a
subset of inbred lines (Zhao et al., 2001; Frame et al.,
2002, 2006, 2011; Ishida et al., 2003; Huang and Wei,
2004; Zhao and Ranch, 2006; Vega et al., 2008).
Microprojectile bombardment was initially used for
transient expression in whole maize tissue (Oard et al.,
1990; Reggiardo et al., 1991; Bansal et al., 1992; Schenk
et al., 1998; Ivanchenko et al., 2000), but the methods
have not yet become routine. Instead, transient gene
expression often relies on transformation of protoplasts isolated from various species (Sheen, 2001;
Davey et al., 2005; Hay and Spanswick, 2007; Horie
et al., 2011; Isayenkov et al., 2011; Jia et al., 2011; Zhou
et al., 2011) or by biolistic transformation of onion
epidermal cells (Scott et al., 1999; Böhlenius et al., 2010;
Wei et al., 2010; Yang et al., 2011; Zhang et al., 2011;
Zou et al., 2011). A particle bombardment system was
optimized in wheat (Triticum aestivum; Schweizer et al.,
1999) and useful for plant pathogen studies in barley
(Hordeum vulgare) and other grasses (Dong et al., 2006),
but has not yet been successfully mimicked in maize.
Transient assays using Agrobacterium infection were
reported for Setaria viridis, an emerging C4 grass
model system (Brutnell et al., 2010), which may effectively replace the distant onion epidermis as a transient model for maize. A direct maize system is still
needed, and experiments in other grasses (Dong et al.,
2006) point to the value of developing a species-specific
transient assay system.
We describe here a reliable approach for transient
expression in maize leaves, permitting more direct
study of protein function and localization at higher efficiency. This system supplements protoplast methods
and replaces the use of dicots or onion epidermis as
platforms for expression, and it allows direct observation of endogenous transient expression in maize. The
method can be used to verify predicted protein expression prior to generating stably transformed lines, to
demonstrate protein-protein colocalization, and to provide for rapid overexpression and dominant-negative
analyses. Transformation efficiency is distinctly increased
due to cell development stage; the method thus has
broad application if researchers take into account the
cellular and developmental context while planning assay
experiments.
RESULTS AND DISCUSSION
Maize leaves and roots grow in a predictable gradient with localized regions of cell division, expansion,
and differentiation (Fig. 1; after Reynolds et al., 1998;
Mitkovski and Sylvester, 2003; Brutnell et al., 2010).
The experiments here were based on the assumption
that successful transient expression would depend on
cellular, developmental, and physiological traits of the
targeted tissues. Cellular features along the leaf gradient suggest that the basal region would be most receptive to successful transformation: epidermal cells at
the base of expanding leaves are densely cytoplasmic,
and they divide and expand rapidly with newly formed
primary cells walls (base region; Fig. 1). Transformation
would be more efficient due to the high proportion of
cytoplasm, early-stage cell walls, and/or the stage of
the cell cycle, which may influence proper transcription
and translation of the transgene. Farther up the leaf
gradient, cells increase in size and contain large central
vacuoles, with the cytoplasm and nucleus compressed
against the more advanced primary cell wall (middle
and tip regions; Fig. 1).
Developing Adult Leaves Are Useful for Transient Assays
Diverse maize tissues were first tested for reliable
transient expression by bombardment. Mature juvenile
and immature adult leaves were compared with root
tips to determine the most effective tissue for establishing a routine procedure. Expression was not observed reliably in nongrowing juvenile leaves or roots
during initial experiments, but further study of these
tissues is warranted. Since expression was consistently
observed after bombardment of still-expanding adult
leaves, we focused our subsequent efforts solely on
this material (for further description, see “Materials
and Methods”). By restricting all transient assays to
the same leaf stage, possible variations in cellular development that may influence receptivity to bombardment were minimized.
Figure 1. Developmental gradients in a maize leaf. A maize leaf (at
left; not to scale) was divided into five regions: two adjacent regions in
the base, a middle region, and two nonadjacent regions at the tip. The
distances from the ligule for each region represent different stages in
the developmental gradient of the leaf (at right), including a site of cell
division immediately above the ligule (left triangle), a gradient of cell
expansion (middle triangle), and a gradient of cell differentiation (right
triangle).
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Transient Transformation of Maize Leaf Cells
Genes Selected for Transient Expression Tests
Several fluorescently labeled proteins (FPs) were
selected to develop optimal transient assays in maize
tissue. The FPs were prioritized based on confirmed
stable expression of the FP in a predicted subcellular
compartment, such as the endoplasmic reticulum (ER),
Golgi, or plasma membrane (Mohanty et al., 2009a,
2009b). Four FPs were generated and used to compare
stable and transient expression in maize: (1) a protein
disulfide isomerase tagged with mCitrine (ZmPDI-YFP),
biochemically localized in the ER; (2) a b-1,2-xylosyltransferase tagged with monomeric Red Fluorescent
Protein (mRFP; ZmXylT-mRFP), predicted to localize
to the Golgi; (3) a b-ketoacyl reductase identified as
GLOSSY8a tagged with mRFP (GL8a-mRFP), biochemically associated with the ER; and (4) a small GTPase
of the Rho-of-Plants family, ZmROP7, tagged with a
monomeric Teal Fluorescent Protein (mTFP; ZmROP7mTFP), predicted to be localized to the plasma membrane
(for further details, see “Materials and Methods”). These
proteins were prioritized because they represent a range
of both well-confirmed (ZmPDI-YFP and GL8a-mRFP)
and predicted (ZmROP7-mTFP and ZmXYLT-mRFP)
localization patterns in diverse membrane compartments, as described more fully here.
The ER compartment was marked by ZmPDI-YFP, a
well-characterized protein common to all eukaryotes,
and GL8a-mRFP, a protein conserved across evolutionarily diverse taxa (Xu et al., 1997). In all systems
studied, including plants (Boston et al., 1996; Gupta and
Tuteja, 2011), PDIs localize to the ER and catalyze reactions associated with disulfide bond formation, promoting the normal folding of substrate proteins. Due to
its conservation of location and function, ZmPDI-YFP
was selected for direct comparison between stable and
transient expression patterns in maize. GL8a-mRFP
functions as a b-ketoacyl reductase responsible for the
biosynthesis of the epicuticular waxes found on maize
leaves and likely other grasses, a function that is restricted to plants, although the protein has homologs in
many eukaryotes (Xu et al., 1997, 2002; Dietrich et al.,
2005). Biochemical evidence has shown an association
between GL8a and the ER (Xu et al., 2002). Thus,
ZmPDI-YFP and GL8a-mFRP can be used to observe
the compartmental localization of distinct ER markers.
The Golgi compartment was marked with ZmXYLTmRFP, a b-1,2-xylosyltransferase that transfers Xyl to
the core of N-linked glycoproteins (Bondili et al., 2006).
The Arabidopsis (Arabidopsis thaliana) homolog,
AtXYLT, which shows 58% identity to ZmXYLT,
localizes to the Golgi in transformed, cultured BY2
tobacco (Nicotiana tabacum) cells (Pagny et al., 2003).
Golgi in plants is a dynamic and multifunctional
organelle system involved in biosynthesis of cell wall
carbohydrates and sorting of cargo in the endomembrane system (Törmäkangas et al., 2001; Neumann
et al., 2003; Jurgens, 2004). Many fundamental questions about plant Golgi function are unanswered, including mechanisms of trafficking for soluble and
membrane proteins and the functional relationship
with the ER (Faso et al., 2009); transient assay methods
will be particularly useful to answer these questions.
A third membrane compartment, the plasma membrane, was observed using ZmROP7, a member of the
plant-specific ROP family of the Ras protein superfamily. ROP proteins have been implicated in pollen
tube growth, cell death, and cell wall synthesis (Delmer
et al., 1995; Lin et al., 1996; Kawasaki et al., 1999; Yang
and Fu, 2007) and were specifically localized to the
plasma membrane in maize cells (Ivanchenko et al., 2000).
A stable line expressing an mTFP-tagged ZmROP7
(ZmROP7-TFP) was recently produced as a color complement to other related proteins, and this reporter line
is now being studied extensively in leaf tissue, where it
shows predicted plasma membrane location of ROP7 in
the leaf, in contrast to prior punctate localization observed in the root (Mohanty et al., 2009a, 2009b). These
constrasting observations in leaves and roots emphasize
that transient expression can be useful for studying
localization differences in diverse tissues.
Efficiency of Transient Transformation Is Highest in the
Base of Immature, Adult Leaves
Transformation efficiency was studied along the leaf
gradient by analyzing transformation success in five
discontinuous segments of expanding adult leaves.
Each leaf was approximately 50 cm in length with a
basal sheath length of only 1 cm or less. The developing
leaf was divided into five parts: two adjacent sectors at
the base, one sector in the middle, and two sectors at the
leaf tip (Fig. 1). Transformation efficiency is an important measure of the reliability of the method and is
defined here as the number of successful transformed
cells per piece of leaf tissue of a given size, in this case
an area of approximately 0.25 cm2. The leaf pieces were
bombarded with polyubiquitin::ZmXylT-mRFP, and
the number of transformed cells was counted 24 h
after bombardment (for details, see “Materials and
Methods”).
Transformation was most efficient in the basal region, 0 to 3 cm from the ligule, and decreased up the
developmental gradient of the leaf, with a nonsignificant increase at 12 to 18 cm from the ligule (Fig. 2A). At
the base, where the transformation frequency is the
highest, a median value of 21.5 transformed cells per
cm2 was calculated, but the values range between 6.25
and 91.67 transformed cells per cm2 (Fig. 2A). As little
as 3 cm farther along the leaf blade, in the region from
3 to 6 cm, transformation dropped to a median value of
less than 1 cell per cm2, a level typical for the remainder of the leaf. Although transformation efficiency
varied, expression was observed throughout the leaf,
albeit at low frequencies in the upper regions (Fig. 2).
Increased transformation efficiency of basal cells can
be explained by their developmental stage: cells in the
base of leaves are smaller and more cytoplasmically
dense with less well-developed primary cell walls and
more rapid cell cycling. To determine if cell size
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Kirienko et al.
cellular differentiation, differences in cell wall structure, and/or stage of the cell cycle.
Transient Expression in Basal and Tip Sectors of a
Developing Maize Leaf
Figure 2. Transformation efficiency in tissue samples from each leaf
region. A, Box plot indicating transformation efficiency per cm2 of
tissue. Leaf region refers to the distance from the ligule, as indicated in
Figure 1. Averages were calculated from two replicates with five tissue
samples per region per replicate. Medians are indicated by thick bars.
The circle above the data represents an outlier data point with a value
more than 1.5 times the inner quartile range. The asterisk indicates a
statistically significant difference from the other groups, as determined
by Student’s t test (P , 0.01). B, Scatterplot graphing the number of
transformants for each piece of tissue versus mean cell area. Each point
represents a single tissue sample. The key indicates the source of each
tissue sample, as described in Figure 1. Arithmetic mean cell area was
calculated from cell sizes from four fields of cells per tissue sample.
correlates with efficiency, cell areas were measured for
each bombarded sample and graphed against the
number of transiently transformed cells (Fig. 2B). The
average cell size varied in these regions, from approximately 260 mm2 in the base to approximately
1,500 mm2 by the middle of the leaf. The correlation
between transformation frequency and leaf blade location was stronger than the correlation between cell
size and transformation frequency (Supplemental Fig.
S1), suggesting that leaf blade position is a better
predictor of transformation efficiency than cell size
alone. This could be explained by other factors such as
Several different constructs were developed, each
tailored to a specific experimental purpose. The polyubiquitin::ZmXYLT-mRFP construct was used to test
whether a strong, constitutive promoter and an efficient
terminator would allow the expression of a complementary DNA (cDNA). All of the other constructs use
native regulatory regions to drive the normal expression of genes of interest. Transient expression of
polyubiquitin::ZmXYLT-mRFP demonstrates a pattern
of small, scattered punctate fluorescence throughout
the cytoplasm, with an increased concentration at the
cell cortex (Fig. 3A) in a pattern consistent with Golgi
colocalization (Satiat-Jeunemaitre et al., 1999). No
other identifiable subcellular compartments were apparent in transiently transformed cells, suggesting that
this nonendogenous promoter/terminator combination
can be used to express this protein as predicted. Figure
3A also shows the use of autofluorescence as a useful
counterpoint to observe transiently expressed fusion
proteins: the punctate red fluorescence of the transgene is highlighted by the blue autofluorescence of the
cell walls. In Figure 3A, the blue autofluorescent guard
cells also contain chloroplasts (normally exhibiting red
autofluorescence) that appear pink due to the blue
wall/red chloroplast merge. Thus, counterstaining of
cell walls, and subsequent effects on cell viability, can
be avoided when autofluorescence can be used to
identify cellular landmarks.
ZmROP7-TFP was bombarded into the base of immature, adult B73 leaves to verify successful transient
expression in additional subcellular compartments
(Fig. 3B). Fluorescence localized to the cell periphery,
consistent with a predicted plasma membrane location
from previous reports (Ivanchenko et al., 2000). Figure
3B highlights the use of bright-field observation in
cases where blue wall autofluorescence may overlap in
wavelength with a fluorophore. In this example, plasma
membrane localization cannot be distinguished from
cytoplasmic, but specific experimental manipulations
such as plasmolysis could be confirmatory.
Transient ZmPDI-YFP Expression Matches
Stable Transformation
One concern with transient expression is the introduction of localization artifacts caused by cell damage
during bombardment or by misexpression. To confirm
normal localization, stable and transient expression
patterns for ZmPDI-YFP were compared. The same
binary vector plasmid used for creating the stably
transformed line was bombarded into nontransgenic
B73 plants, and expression was compared with its
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Transient Transformation of Maize Leaf Cells
Figure 3. Transient and stable expression patterns of FPs. Confocal laser scanning microscopy images show transient expression
of ZmXYLT-mRFP (A), transient expression of ZmROP7-mTFP (B), and stable (C) and transient (D–F) expression of ZmPDI-YFP in
adaxial epidermal cells. Maximum intensity projections of confocal stacks (A, C, and E) show all confocal sections collected
from a cell. In A, cell wall autofluorescence is blue and the merge of chlorophyll and cell wall autofluorescence in guard cells
appears pink. A single transformed cell (D–F) has been separated into only cortical planes (D), a maximum projection (E), and a
merge of cortical and medial sections (F). In F, differential pseudocoloring of medial ZmPDI-YFP (magenta) and cortical ZmPDIYFP (green) are used to enhance visualization. Bars = 10 mm.
comparable, stably transformed line. A maximum
projection through a partial scan of blade epidermal
cells of the stable transformant is shown for comparison (Fig. 3C; as this is a partial scan, the surfaces
of all cells are not shown). Here, ZmPDI-YFP appears
as a reticulate network localized in the cortical region
of the cell (Fig. 3C), consistent with observations of
ER structure in plant cells (Satiat-Jeunemaitre et al.,
1999). In transiently transformed cells, ZmPDI-YFP
also appears as a reticulate network in the cortex of
adaxial epidermal cells (Fig. 3D). A maximum intensity projection through 16 Z planes of a cell
demonstrates that this reticulate pattern is pronounced in the cell cortex (Fig. 3D). In the noncortical
regions of the cell, expression is noted in cytoplasmic strands and the perinuclear region (Fig. 3E, arrow). Cell damage and/or autofluorescence are not
detected in neighboring cells. The similarity of the
fluorescence patterns in stably and transiently
transformed cells suggests, for this ER marker protein, that localization was not affected by transient
expression.
The maximum intensity projections shown in Figure 3E are useful for simultaneously viewing fluorescence patterns across multiple Z planes. However,
localization of the same protein to more than one
subcellular region can complicate the interpretation
of fluorescence patterns. For example, in Figure 3E,
the cortical, reticulate network and the perinuclear
and cytoplasmic fluorescence patterns for ZmPDIYFP are not readily distinguishable. To simplify the
visualization of these separate patterns, fluorescence
from the same FP, ZmPDI-YFP, was differentially
false colored, based on Z location. For example,
medial Z planes were colored magenta, while cortical planes near the epidermal surface were shaded
green. Thus, the three-dimensional depth of ZmPDIYFP is emphasized while still being represented in
a single image (Fig. 3F). The imaging technique
makes it clear that the cortical ZmPDI-YFP at nonepidermal cell surfaces demonstrates a reticulate
nature similar to that at the epidermal surface (Fig. 3,
compare E and F). Imaging methods such as these
optimize viewing of the three-dimensionally rendered
object.
Transient Cotransformation to Localize Multiple Protiens
Simultaneous localization of multiple proteins can
be used to study protein function and to test for potential interactions. Transient transformation of two
proteins together can make the experimental study
more efficient. Coexpression from two separate vectors
can be accomplished in two ways: first, two or more
tagged proteins can be simultaneously transiently
expressed by cobombardment of their encoding plasmids; second, a tagged protein can be transiently
expressed in a stably transformed maize line expressing a fusion protein.
ZmXYLT-mRFP and ZmPDI-YFP were coprecipitated onto microcarriers and bombarded together into
tissue sections from the base of a growing adult B73
leaf (Fig. 4, A–C). ZmPDI-YFP demonstrates the ERspecific reticulate pattern previously observed (compare Fig. 3, C and D, with Fig. 4A). ZmXYLT-mRFP
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Kirienko et al.
shows a punctate distribution concentrated at the
periphery of the cell, as was described previously
(compare Figs. 3A and 4B). The merged image demonstrates that the two proteins are transiently coexpressed in the same cell (Fig. 4C). Notably, the
localization of each transiently expressed protein remains independent and apparently correct, reinforcing the conclusion that localization patterns are
not altered during simultaneous expression of more
than one construct.
Transient Expression in a Stably Expressed Line for
Colocalization Study
An important application for a reliable transient
expression system is to localize proteins rapidly in a
plant that reproduces slowly, such as maize. In addition to the cobombardment, it would be an advantage
to colocalize transiently expressed proteins in already
stable lines. The stably transformed GL8a-RFP ER
marker line confirmed the feasibility of this approach
(Fig. 4, D–F). The tagged protein was first stably expressed in maize (Fig. 4E) using methods described
previously (Mohanty et al., 2009a, 2009b). A second ER
marker, ZmPDI-YFP, as described above, was transiently expressed in the GL8a-mRFP marker line to
coexpress two ER markers in the same plant and to
test for colocalization. Transiently expressed ZmPDIYFP showed the expected cortical reticulate networks
and perinuclear fluorescence (Fig. 4D). Interestingly,
GL8a-mRFP localized in a diffuse pattern through the
cytoplasm, with minimal reticulation into networks
(Fig. 4, E and F). There are at least two explanations to
explain the localization pattern of GL8a-mRFP. First,
protein localization may be different during adult
leaf development because GL8a functions when juvenile epicuticular wax is being synthesized (Bianchi
et al., 1985; Xu et al., 1997). Second, GL8a and ZmPDI
may localize to different ER subcompartments, a
phenomenon that has been described previously
(Staehelin, 1997). Notably, none of the coexpression
analyses performed using bombardment have exhibited signs of mislocalization when compared with stably transformed lines.
Further evaluation of the ER localization patterns of
the two markers suggests colocalization in the perinuclear
and dispersed cortical regions (merging to white or
lime-green regions in Fig. 4F). The merged image also
shows that the reticulate network at the adaxial epidermal surface shows only ZmPDI-YFP (Fig. 4F). The
partial colocalization suggests that the two proteins
function or are transported through both distinct and
overlapping domains of the ER, but additional work
will verify this possibility. These observations support
the subcompartmentation hypothesis for the ER and
point to the value of using transient bombardment in
plant lines that have already been engineered to stably
express an FP.
The observation of partial colocalization is intriguing.
Further study is needed and can be accomplished by
transient expression assays. For example, once GL8aRFP is proven to be localized and functioning correctly,
by complementation tests with a gl8a mutant, further
experiments could test whether GL8a-mRFP localizes in
a distinct ER domain. This could be a consequence of
different biological functions. GL8a is an enzyme involved in wax synthesis (Xu et al., 1997, 2002), whereas
PDI is predicted to be a protein chaperone, as in other
systems (Boston et al., 1996; Li and Larkins, 1996; Gupta
and Tuteja, 2011). Thus, the localization data would
clarify if unique ER domains are associated with verylong-chain fatty acids. Up to 16 different functional regions of the ER have been predicted in plants (Staehelin,
1997), and the extent of functional overlap may be resolved in vivo using transient assay methods.
CONCLUSION
Microprojectile bombardment for transient expression assays in maize has been reported previously, but
Figure 4. A to C, Examples of transient
coexpression. ZmPDI-YFP (green; A)
and ZmXylT-mRFP (magenta; B) show
minimal colocalization in the merged
image (C). In this example, only cortical
expression of ZmPDI-YFP is shown. D to
F, Transient coexpression of ZmPDI-YFP
(green; D) in a cell that is also stably
expressing GL8a-mRFP (magenta; E)
identifies partial colocalization in the
merged image (F). Bars = 10 mm.
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Transient Transformation of Maize Leaf Cells
the methods are not yet routine. Prior attempts may
have used only the easily accessible leaf middle and tip
regions, where transformation efficiency is low, averaging
less than one transformed cell per cm2 (Fig. 2A). Using
the more efficient basal tissues, transformation success
is greatly improved. Differences in transformation efficiency may be due to the developmental state of the
cell, which influences various factors including cell
wall composition and cell division/cell expansion
profiles. For example, wall lignification begins approximately 8 to 14 cm from the ligule, increasing the
strength and the rigidity of the cell walls (Vincent
et al., 2005). Alternatively, comparatively higher levels
of b1,3-b1,4 mixed linkage glucans are found in base
tissue (Carpita et al., 2001). The hydrolysis of these
compounds is necessary for cell growth (Hoson et al.,
1992; Inouhe and Nevins, 1998); these wall features are
indicators of the developmental state of cells.
Basal leaf tissue is ideal for the optimal expression of
proteins of standard or constitutive expression levels,
as shown here. Proteins expressed at low levels, in
very specific subsets of cell types, or at distinct phases
of the cell cycle would require selected tissue sampling. Transient expression is successful either using a
gene’s endogenous promoter, terminator, and introns
or a strong promoter-terminator combination to drive
a cDNA construct, as in the case of ZmXYLT-mRFP.
Previous reports show that transient expression in
cultured maize cells is improved using an intron for
high levels of expression (Callis et al., 1987): the 59
untranslated region of the polyubiquitin gene contains
an endogenous intron (Christensen et al., 1992), making this promoter ideal for expressing cDNA sequences via microprojectile bombardment in the intact
maize leaf. Promoter studies are also feasible, by engineering a minimal expression cassette similar to that
developed for ZmXYLT-mRFP, but with the entire
coding region for the gene of interest. Placing a multiple cloning site upstream of the start codon would
facilitate promoter swaps and truncations, yielding
information on promoter function in maize and other
grasses. Gateway cloning, as used here for ZmPDIYFP and ZmROP7-mTFP, further increases versatility.
Although the overexpression of secreted proteins can
alter localization patterns (Brandizzi et al., 2003;
Lisenbee et al., 2003), particularly if sorting pathways
are saturable, ZmXYLT-mRFP appeared to be localized
as predicted.
We report here a reliable method for robust and
efficient transient expression of fluorescently tagged
proteins in intact maize leaf cells of the correct age and
size. The method can be used to express genes with
their native regulatory sequences, including introns, or
as cDNAs using a constitutive promoter and a heterologous terminator. Transient expression is consistent
with observations of stably transformed lines and can
be used for localization, colocalization, and functional
studies. These approaches will complement newly
emerging capabilities for functional genomics studies
in maize (Mohanty et al., 2009a, 2009b; Penning et al.,
2009; Li et al., 2010; Sekhon et al., 2011; Cook et al.,
2012). The reliability and utility of the transient assay
methods described here are thus essential to exploit
fully the maize genome for systems biology study, and
the focus on developmental state will have broad
application to other plants that may be resistant to
transient expression.
MATERIALS AND METHODS
Plant Growth Procedures
Maize (Zea mays) plants from the B73 inbred line were used for transient
procedures that did not involve stably transformed lines. For experiments involving stably transformed lines, maize transformation was accomplished by the
Plant Transformation Facility at Iowa State University using Agrobacterium
tumefaciens-mediated transformation. T0 seedlings were outcrossed at least two
generations into a B73 inbred line. Single and stable transgene inserts were
confirmed by 50% segregation of the transgene in T1 and T2 generation progeny.
Inbred and transgenic seeds were planted in standard fertilized potting soil and
grown in a controlled environment at 25°C with a 16-h-light/8-h-dark photoperiod for 2 to 4 weeks to the appropriate L9 stage and L3 stages.
Plasmid Construction
pUBI-1-XylTmRFP-Vsp encodes a C-terminally, mRFP-tagged, Golgi-resident
b-1,2-xylosyltransferase whose expression is driven by a maize polyubiquitin
promoter fragment (Christensen et al., 1992) with the terminator from soybean
(Glycine max) vegetative storage protein (Mason et al., 1988). This plasmid was
created as follows. pUC-mRFP was generated by inserting a PCR-amplified
mRFP into the XbaI and SphI sites of pUC19. pUC-XylTmRFP was generated
by subcloning a PCR-amplified b-1,2-xylosyltransferase (GRMZm2G419267;
Bondili et al., 2006) open reading frame from B73 cDNA into the KpnI and XbaI
sites of pUC-mRFP. pUC-Vsp was created by inserting a PCR-amplified
soybean Vsp terminator sequence from pTF101.1 (generously provided by Dr.
Kan Wang) into the SphI and HindIII sites of pUC19. pUBI-1-Vsp was generated by subcloning the PCR-amplified polyubiquitin promoter from
pAHC25 (generously provided by Dr. Kan Wang) into the SacI and KpnI sites
of pUC-Vsp. The final construct, pUBI1-XylTmRFP-Vsp, was generated by
inserting a KpnI-SphI fragment from pUC-XylTmRFP into pUBI-1-Vsp.
pTF101.1-gl8a-mRFP was generated by triple-template PCR methods as described previously (Mohanty et al., 2009a, 2009b) using GL8a (Xu et al., 1997)
and the mRFP fluorescent protein genes (Campbell et al., 2002). pTF101.1ZmPDI-YFP was generated using a MultiSite Gateway three-way cloning
method (Invitrogen) with the ZmPDIL1-1 (GRMZm2G091481; Houston, et al.,
2005) and mCitrine fluorescent protein genes (Heikal et al., 2000), as was
pTF101.1-ZmROP7-mTFP, which used a modified cyan fluorescent protein
derivative (Ai et al., 2006). Additional details and protocols can be found at
http://maize.jcvi.org/cellgenomics/protocol.shtml.
All plasmids were sequence verified after steps involving PCR or by restriction mapping after steps involving ligation or propagation. All final
constructs were sequence verified.
Preparation of Tissue Samples for
Microprojectile Bombardment
Approximately 0.25-cm2 tissue samples were cut from plant leaves for each
experiment. With the exception of experiments testing differences in transformation efficiency based on tissue maturity, all tissue tested was within 3 cm
of the ligule and not yet greened. Tissue was obtained as follows. First, freshly
grown, age-appropriate (between six and 13 leaves emerged), greenhousegrown plants were cut transversely through the stalk at the level of the soil.
Outer leaves were sequentially removed until the final emergent leaf was
discarded. Once nonemergent leaves were obtained, the first of these leaves
was generally discarded and the subsequent nonemergent leaf was selected
for use, based on the position of the ligule. Attention was focused on a developing
leaf that contained 1 cm of sheath tissue and a partly developed ligule fringe.
The 1 cm of sheath tissue was discarded. Material within 3 cm of the developing ligule was preferentially used for all assays, except in quantitative
Plant Physiol. Vol. 159, 2012
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Kirienko et al.
experiments testing for the efficiency of transformation as a function of age.
Dissections were conducted in a small pool of water to prevent dehydration of
the tissue. After being cut into approximately 0.25-cm2 pieces, leaf samples
were distributed densely, adaxial side up, on Murashige and Skoog agar plates
(4.4%, w/v; Sigma-Aldrich). Plates were bombarded within 1 h of dissection.
For experiments testing the effect of developmental stage on transformation
efficiency, the same procedure was used except that samples of 0.25 cm2 were
cut at the positions described in Figure 1. Tissue was separated by developmental stage, placed on Murashige and Skoog agar plates, and bombarded
within 1 h of dissection. Experiments using particular cell types for transformation would require harvesting tissue from age-appropriate times and locations.
Statistical Computing). Regression values were calculated using a least-squares
method in Microsoft Excel. Student’s t tests were performed on transformation
frequency data using R, and outliers were defined as data greater than 1.5
times the inner quartile range as determined by the graphing function.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Tissue location correlates with transformation
efficiency.
Supplemental Figure S2. Transformation efficiency is independent of
DNA purification method.
Microprojectile Bombardment
DNA was prepared by commercially available column purification in either
miniprep or maxiprep formats (Qiagen) or by classical alkaline lysis followed by
cesium chloride-ethidium bromide isopycnic gradient purification (Sambrook
et al., 1989). Equal concentrations of DNA, as determined by both spectroscopy and analytical gels, showed no difference in transformation efficiency
(Supplemental Fig. S2).
All bombardments were performed with Bio-Rad Tungsten M-10 microcarriers
using 1 mg of DNA precipitated onto 166 mg of tungsten microcarriers (approximately 5 3 107 microcarriers) per bombardment. Procedures, with slight modifications to the manufacturer’s instructions, were as follows. First, 20 mg of
microcarriers was measured in 1.5-mL microcentrifuge tubes, washed for 5 min
with vigorous mixing in 1 mL of 70% ethanol, soaked for 15 min, and then pelleted
by microcentrifugation for 10 s. The pelleted microcarriers were washed for 1 min
with vigorous mixing in 1 mL of sterile water three times and then resuspended in
1 mL of sterile 50% glycerol. Microcarriers were stored at 220°C until use.
Prepared microcarriers were vortexed vigorously for 5 min prior to dividing into
aliquots to break up any agglomerates; 50 mL of prepared microcarriers was then
transferred to a fresh microcentrifuge tube and mixed continuously using a platform vortexer with the following added sequentially: 6 mL of DNA at a concentration of 1.0 mg mL21 and 50 mL of 2.5 M CaCl2, with each addition mixed for
1 min. Finally, 20 mL of 0.1 M spermidine was added and allowed to mix for 5 min.
Microcarriers and DNA were centrifuged for 15 s, and the pellet was
resuspended and washed in 140 mL of 70% HPLC-grade ethanol twice using
100% HPLC-grade ethanol during the final stage. The microcarriers were
resuspended in 50 mL of 100% HPLC-grade ethanol, and 8 mL was transferred
to macrocarriers (Bio-Rad) for subsequent bombardment. Macrocarriers were
covered to prevent the accumulation of dust while ethanol evaporated and
then used for bombardment.
All bombardments were performed with a PDS-1000/He system (Bio-Rad).
Rupture discs (1,350 p.s.i.; Bio-Rad) were used to build system pressure. Tissue
samples were situated 9 cm from the macrocarrier stopping screen, and no other
sample screen was used. Helium pressure at the tank was regulated at 1,600 p.s.i.
Vacuum pressure within the chamber was allowed to reach at least 20 inches of
mercury prior to firing. After bombardment, tissue was allowed to recover for
16 to 24 h at 22°C in the dark (to limit tissue greening) prior to analysis.
Confocal Microscopy and Image Analysis
Bombarded tissue samples were mounted in water and imaged using a 633 1.4
numerical aperture objective on a Zeiss LSM710 microscope using 514- and 561nm laser lines with ZEN 2010 software (Zeiss). Normal emission spectra of FPs
and autofluorescence were narrowed using the ZEN 2010 software function to
autoselect emission range, thus avoiding overlapping emission. The normal
emission of cell wall autofluorescence ranges between 350 and 550 nm. This
spectrum was narrowed to 425 to 500 nm to avoid coemission with the lower
emitting wavelengths of mYFP, which normally emits from 500 to 625 nm. The
mYFP fluorescence emission spectrum was also narrowed to 525 to 560 nm to
ensure no overlap with the cell wall. mTFP emission, normally from 450 to 600 nm,
was narrowed to avoid cell wall autofluorescence. The normal emission spectrum
for mRFP is from 560 to 750 nm. Chlorophyll autofluorescence, which normally
emits between 620 and 800 nm, was avoided by narrowing the emission for mRFP
to 570 to 615 nm. Images were analyzed and manipulated using MetaMorph
Software (Molecular Devices) or ImageJ software (National Institutes of Health).
Data Analysis
Statistical analysis and final data presentation were prepared using
Microsoft Excel and the R Statistical Programming Language (R Foundation for
ACKNOWLEDGMENTS
We thank Kan Wang, Bronwyn Frame, and the Plant Transformation
Facility at Iowa State University for stable maize transformation and Zhaojie
Zhang for assistance at the University of Wyoming Core Microscope Facility.
We thank Jay Gatlin for helpful discussions on data analysis and visualization
and Carolyn Rasmussen and Natalia Kirienko for providing helpful comments
on the manuscript.
Received May 3, 2012; accepted June 7, 2012; published June 15, 2012.
LITERATURE CITED
Ai HW, Henderson JN, Remington SJ, Campbell RE (2006) Directed evolution of a monomeric, bright and photostable version of Clavularia
cyan fluorescent protein: structural characterization and applications in
fluorescence imaging. Biochem J 400: 531–540
Amien S, Kliwer I, Márton ML, Debener T, Geiger D, Becker D,
Dresselhaus T (2010) Defensin-like ZmES4 mediates pollen tube burst
in maize via opening of the potassium channel KZM1. PLoS Biol 8:
e1000388
Bansal KC, Viret JF, Haley J, Khan BM, Schantz R, Bogorad L (1992)
Transient expression from cab-m1 and rbcS-m3 promoter sequences is
different in mesophyll and bundle sheath cells in maize leaves. Proc Natl
Acad Sci USA 89: 3654–3658
Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium mediated
gene transfer by infiltration of adult Arabidopsis thaliana plants. C R
Acad Sci Paris Life Sci 316: 1194–1199
Bevan MW, Flavell RB, Chilton M-D (1983) A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature
304: 184–187
Bianchi A, Bianchi G, Avato P, Salamini F (1985) Biosynthetic pathways of
epicuticular wax of maize as assessed by mutation, light, plant age, and
inhibitor studies. Maydica 30: 179–198
Böhlenius H, Mørch SM, Godfrey D, Nielsen ME, Thordal-Christensen H
(2010) The multivesicular body-localized GTPase ARFA1b/1c is important for callose deposition and ROR2 syntaxin-dependent preinvasive basal defense in barley. Plant Cell 22: 3831–3844
Bondili JS, Castilho A, Mach L, Glössl J, Steinkellner H, Altmann F,
Strasser R (2006) Molecular cloning and heterologous expression of
beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from
maize. Phytochemistry 67: 2215–2224
Boston RS, Viitanen PV, Vierling E (1996) Molecular chaperones and
protein folding in plants. Plant Mol Biol 32: 191–222
Brandizzi F, Hanton S, DaSilva LL, Boevink P, Evans D, Oparka K,
Denecke J, Hawes C (2003) ER quality control can lead to retrograde
transport from the ER lumen to the cytosol and the nucleoplasm in
plants. Plant J 34: 269–281
Brutnell TP, Wang L, Swartwood K, Goldschmidt A, Jackson D, Zhu XG,
Kellogg E, Van Eck J (2010) Setaria viridis: a model for C4 photosynthesis. Plant Cell 22: 2537–2544
Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in
cultured maize cells. Genes Dev 1: 1183–1200
Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias
DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl
Acad Sci USA 99: 7877–7882
Plant Physiol. Vol. 159, 2012
1316
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Transient Transformation of Maize Leaf Cells
Carpita NC, Defernez M, Findlay K, Wells B, Shoue DA, Catchpole G,
Wilson RH, McCann MC (2001) Cell wall architecture of the elongating
maize coleoptile. Plant Physiol 127: 551–565
Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner
TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115: 971–980
Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin
genes: structure, thermal perturbation of expression and transcript
splicing, and promoter activity following transfer to protoplasts by
electroporation. Plant Mol Biol 18: 675–689
Cook JP, McMullen MD, Holland JB, Tian F, Bradbury P, Ross-Ibarra J,
Buckler ES, Flint-Garcia SA (2012) Genetic architecture of maize kernel
composition in the nested association mapping and inbred association
panels. Plant Physiol 158: 824–834
Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts:
status and biotechnological perspectives. Biotechnol Adv 23: 131–171
De Block M, Herrera-Estrella L, Van Montagu M, Schell J, Zambryski P
(1984) Expression of foreign genes in regenerated plants and in their
progeny. EMBO J 3: 1681–1689
Delmer DP, Pear JR, Andrawis A, Stalker DM (1995) Genes encoding
small GTP-binding proteins analogous to mammalian rac are
preferentially expressed in developing cotton fibers. Mol Gen Genet 248:
43–51
Dietrich CR, Perera MA, D Yandeau-Nelson M, Meeley RB, Nikolau BJ,
Schnable PS (2005) Characterization of two GL8 paralogs reveals that
the 3-ketoacyl reductase component of fatty acid elongase is essential for
maize (Zea mays L.) development. Plant J 42: 844–861
Dong W, Nowara D, Schweizer P (2006) Protein polyubiquitination plays a
role in basal host resistance of barley. Plant Cell 18: 3321–3331
Eveland AL, Satoh-Nagasawa N, Goldshmidt A, Meyer S, Beatty M,
Sakai H, Ware D, Jackson D (2010) Digital gene expression signatures
for maize development. Plant Physiol 154: 1024–1039
Faso C, Boulaflous A, Brandizzi F (2009) The plant Golgi apparatus: last 10
years of answered and open questions. FEBS Lett 583: 3752–3757
Frame B, Main M, Schick R, Wang K (2011) Genetic transformation using
maize immature zygotic embryos. Methods Mol Biol 710: 327–341
Frame BR, McMurray JM, Fonger TM, Main ML, Taylor KW, Torney FJ,
Paz MM, Wang K (2006) Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep 25:
1024–1034
Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg
SE, Li B, Nettleton DS, Pei D, et al (2002) Agrobacterium tumefaciensmediated transformation of maize embryos using a standard binary
vector system. Plant Physiol 129: 13–22
Gore MA, Chia JM, Elshire RJ, Sun Q, Ersoz ES, Hurwitz BL, Peiffer JA,
McMullen MD, Grills GS, Ross-Ibarra J, et al (2009) A first-generation
haplotype map of maize. Science 326: 1115–1117
Gupta D, Tuteja N (2011) Chaperones and foldases in endoplasmic reticulum stress signaling in plants. Plant Signal Behav 6: 232–236
Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF, Zhang JZ
(2002) Transcription factor CBF4 is a regulator of drought adaptation in
Arabidopsis. Plant Physiol 130: 639–648
Hay JO, Spanswick RM (2007) Isolation of ripening rice (Oryza sativa L.)
aleurone protoplasts for uptake studies. Plant Sci 172: 659–664
Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW (2000) Molecular
spectroscopy and dynamics of intrinsically fluorescent proteins: coral
red (dsRed) and yellow (Citrine). Proc Natl Acad Sci USA 97: 11996–
12001
Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of
rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis
of the boundaries of the T-DNA. Plant J 6: 271–282
Horie T, Brodsky DE, Costa A, Kaneko T, Lo Schiavo F, Katsuhara M,
Schroeder JI (2011) K+ transport by the OsHKT2;4 transporter from rice
with atypical Na+ transport properties and competition in permeation of
K+ over Mg2+ and Ca2+ ions. Plant Physiol 156: 1493–1507
Horsch RB, Fraley RT, Rogers SG, Sanders PR, Lloyd A, Hoffmann N (1984)
Inheritance of functional foreign genes in plants. Science 223: 496–498
Hoson T, Masuda Y, Nevins DJ (1992) Comparison of the outer and inner
epidermis: inhibition of auxin-induced elongation of maize coleoptiles
by glucan antibodies. Plant Physiol 98: 1298–1303
Houston NL, Fan C, Xiang QY, Schulze JM, Jung R, Boston RS (2005)
Phylogenetic analyses identify 10 classes of the protein disulfide
isomerase family in plants, including single-domain protein disulfide
isomerase-related proteins. Plant Physiol 137: 762–778
Huang XQ, Wei ZM (2004) High-frequency plant regeneration through
callus initiation from mature embryos of maize (Zea mays L.). Plant Cell
Rep 22: 793–800
Inouhe M, Nevins DJ (1998) Changes in the activities and polypeptide
levels of exo- and endoglucanases in cell walls during developmental
growth of Zea mays coleoptiles. Plant Cell Physiol 39: 762–768
Isayenkov S, Isner JC, Maathuis FJ (2011) Rice two-pore K+ channels are
expressed in different types of vacuoles. Plant Cell 23: 756–768
Ishida Y, Saito H, Hiei Y, Komari T (2003) Improved protocol for transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Plant Biotechnol 21: 57–63
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High
efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14: 745–750
Ivanchenko M, Vejlupkova Z, Quatrano RS, Fowler JE (2000) Maize ROP7
GTPase contains a unique, CaaX box-independent plasma membrane
targeting signal. Plant J 24: 79–90
Jia H, Ren H, Gu M, Zhao J, Sun S, Zhang X, Chen J, Wu P, Xu G (2011)
The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice. Plant Physiol 156: 1164–1175
Jurgens G (2004) Membrane trafficking in plants. Annu Rev Cell Dev Biol
20: 481–504
Kawasaki T, Henmi K, Ono E, Hatakeyama S, Iwano M, Satoh H,
Shimamoto K (1999) The small GTP-binding protein rac is a regulator of
cell death in plants. Proc Natl Acad Sci USA 96: 10922–10926
Klein TM, Gradziel TM, Fromm ME, Sanford JC (1988) Factors influencing gene delivery in Zea mays cells by high velocity microprojectiles.
Nat Biotechnol 6: 559–563
Kumpatla SP, Teng W, Buchholz WG, Hall TC (1997) Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice. Plant Physiol 115: 361–373
Li CP, Larkins BA (1996) Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant. Plant Mol Biol 30:
873–882
Li P, Ponnala L, Gandotra N, Wang L, Si Y, Tausta SL, Kebrom TH,
Provart N, Patel R, Myers CR, et al (2010) The developmental dynamics
of the maize leaf transcriptome. Nat Genet 42: 1060–1067
Lin Y, Wang Y, Zhu JK, Yang Z (1996) Localization of a Rho GTPase implies a role in tip growth and movement of the generative cell in pollen
tubes. Plant Cell 8: 293–303
Lisenbee CS, Karnik SK, Trelease RN (2003) Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal
ER. Traffic 4: 491–501
Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB
(1986) Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens. Science 234: 464–466
Lu C, Han MH, Guevara-Garcia A, Fedoroff NV (2002) Mitogen-activated
protein kinase signaling in postgermination arrest of development by
abscisic acid. Proc Natl Acad Sci USA 99: 15812–15817
Manickavasagam M, Ganapathi A, Anbazhagan VR, Sudhakar B,
Selvaraj N, Vasudevan A, Kasthurirengan S (2004) Agrobacteriummediated genetic transformation and development of herbicide-resistant
sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell
Rep 23: 134–143
Mason HS, Guerrero FD, Boyer JS, Mullet JE (1988) Proteins homologous
to leaf glycoproteins are abundant in stems of dark-grown soybean
seedlings: analysis of proteins and cDNAs. Plant Mol Biol 11: 845–856
Mitkovski M, Sylvester AW (2003) Analysis of cell patterns in developing
maize leaves: dark-induced cell expansion restores normal division orientation in the mutant tangled. Int J Plant Sci 164: 113–124
Mohanty A, Luo A, DeBlasio S, Ling X, Yang Y, Tuthill DE, Williams KE,
Hill D, Zadrozny T, Chan A, et al (2009a) Advancing cell biology and
functional genomics in maize using fluorescent protein-tagged lines.
Plant Physiol 149: 601–605
Mohanty A, Yang Y, Luo A, Sylvester AW, Jackson D (2009b) Methods for
generation and analysis of fluorescent protein-tagged maize lines.
Methods Mol Biol 526: 71–89
Neumann U, Brandizzi F, Hawes C (2003) Protein transport in plant cells:
in and out of the Golgi. Ann Bot (Lond) 92: 167–180
Plant Physiol. Vol. 159, 2012
1317
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Kirienko et al.
Oard JH, Paige DF, Simmonds JA, Gradziel TM (1990) Transient gene
expression in maize, rice, and wheat cells using an airgun apparatus.
Plant Physiol 92: 334–339
Pagny S, Bouissonnie F, Sarkar M, Follet-Gueye ML, Driouich A,
Schachter H, Faye L, Gomord V (2003) Structural requirements for
Arabidopsis beta1,2-xylosyltransferase activity and targeting to the
Golgi. Plant J 33: 189–203
Penning BW, Hunter CT III, Tayengwa R, Eveland AL, Dugard CK, Olek
AT, Vermerris W, Koch KE, McCarty DR, Davis MF, et al (2009) Genetic resources for maize cell wall biology. Plant Physiol 151: 1703–1728
Reggiardo MI, Arana JL, Orsaria LM, Permingeat HR, Spitteler MA,
Vallejos RH (1991) Transient transformation of maize tissues by microprojectile bombardment. Plant Sci 75: 237–243
Reynolds JO, Eisses JF, Sylvester AW (1998) Balancing division and expansion during maize leaf morphogenesis: analysis of the mutant,
warty-1. Development 125: 259–268
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY
Satiat-Jeunemaitre B, Boevink P, Hawes C (1999) Membrane trafficking in
higher plant cells: GFP and antibodies, partners for probing the secretory pathway. Biochimie 81: 597–605
Schenk PM, Elliott AR, Manners JM (1998) Assessment of transient gene
expression in plant tissues using the green fluorescent protein. Plant Mol
Biol Rep 16: 313–322
Schnable JC, Springer NM, Freeling M (2011) Differentiation of the maize
subgenomes by genome dominance and both ancient and ongoing gene
loss. Proc Natl Acad Sci USA 108: 4069–4074
Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C,
Zhang J, Fulton L, Graves TA, et al (2009) The B73 maize genome:
complexity, diversity, and dynamics. Science 326: 1112–1115
Scott A, Wyatt S, Tsou PL, Robertson D, Allen NS (1999) Model system for
plant cell biology: GFP imaging in living onion epidermal cells. Biotechniques 26: 1125–1132
Schweizer P, Pokorny J, Abderhalden O, Dudler R (1999) A transient
assay system for the functional assessment of defense-related genes in
wheat. Mol Plant Microbe Interact 12: 647–654
Sekhon RS, Lin H, Childs KL, Hansey CN, Buell CR, de Leon N,
Kaeppler SM (2011) Genome-wide atlas of transcription during maize
development. Plant J 66: 553–563
Sheahan MB, Staiger CJ, Rose RJ, McCurdy DW (2004) A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin
highlights new features of a dynamic actin cytoskeleton in live plant
cells. Plant Physiol 136: 3968–3978
Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll
protoplasts. Plant Physiol 127: 1466–1475
Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8: 291–298
Songstad DD, Somers DA, Griesbach RJ (1995) Advances in alternative
DNA delivery techniques. Plant Cell Tissue Organ Cult 40: 1–15
Springer NM, Ying K, Fu Y, Ji T, Yeh CT, Jia Y, Wu W, Richmond T,
Kitzman J, Rosenbaum H, et al (2009) Maize inbreds exhibit high levels
of copy number variation (CNV) and presence/absence variation (PAV)
in genome content. PLoS Genet 5: e1000734
Staehelin LA (1997) The plant ER: a dynamic organelle composed of a large
number of discrete functional domains. Plant J 11: 1151–1165
Swanson-Wagner RA, DeCook R, Jia Y, Bancroft T, Ji T, Zhao X, Nettleton D,
Schnable PS (2009) Paternal dominance of trans-eQTL influences gene expression patterns in maize hybrids. Science 326: 1118–1120
Tingay S, McElroy D, Kalla R, Fieg SJ, Wang M-B, Thornton S, Brettell R
(1997) Agrobacterium tumefaciens-mediated barley transformation.
Plant J 11: 1369–1376
Törmäkangas K, Hadlington JL, Pimpl P, Hillmer S, Brandizzi F, Teeri
TH, Denecke J (2001) A vacuolar sorting domain may also influence the
way in which proteins leave the endoplasmic reticulum. Plant Cell 13:
2021–2032
Tsuda K, Qi Y, Nguyen LV, Bethke G, Tsuda Y, Glazebrook J, Katagiri F
(2012) An efficient Agrobacterium-mediated transient transformation of
Arabidopsis. Plant J 69: 713–719
Vasil IK (1994) Molecular improvement of cereals. Plant Mol Biol 25: 925–937
Vega JM, Yu W, Kennon AR, Chen X, Zhang ZJ (2008) Improvement of
Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27: 297–305
Vincent D, Lapierre C, Pollet B, Cornic G, Negroni L, Zivy M (2005) Water
deficits affect caffeate O-methyltransferase, lignification, and related
enzymes in maize leaves: a proteomic investigation. Plant Physiol 137:
949–960
Wei X, Xu J, Guo H, Jiang L, Chen S, Yu C, Zhou Z, Hu P, Zhai H, Wan J
(2010) DTH8 suppresses flowering in rice, influencing plant height and
yield potential simultaneously. Plant Physiol 153: 1747–1758
Wullems GJ, Molendijk L, Ooms G, Schilperoort RA (1981) Differential
expression of crown gall tumor markers in transformants obtained after
in vitro Agrobacterium tumefaciens-induced transformation of cell wall
regenerating protoplasts derived from Nicotiana tabacum. Proc Natl
Acad Sci USA 78: 4344–4348
Xu X, Dietrich CR, Delledonne M, Xia Y, Wen TJ, Robertson DS, Nikolau
BJ, Schnable PS (1997) Sequence analysis of the cloned glossy8 gene of
maize suggests that it may code for a b-ketoacyl reductase required for
the biosynthesis of cuticular waxes. Plant Physiol 115: 501–510
Xu X, Dietrich CR, Lessire R, Nikolau BJ, Schnable PS (2002) The endoplasmic reticulum-associated maize GL8 protein is a component of the
acyl-coenzyme A elongase involved in the production of cuticular
waxes. Plant Physiol 128: 924–934
Yang X, Yang YN, Xue LJ, Zou MJ, Liu JY, Chen F, Xue HW (2011) Rice
ABI5-Like1 regulates abscisic acid and auxin responses by affecting the
expression of ABRE-containing genes. Plant Physiol 156: 1397–1409
Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22: 543–551
Yang Z, Fu Y (2007) ROP/RAC GTPase signaling. Curr Opin Plant Biol 10:
490–494
Zhang Z, Zhang Y, Tan H, Wang Y, Li G, Liang W, Yuan Z, Hu J, Ren H,
Zhang D (2011) RICE MORPHOLOGY DETERMINANT encodes the type II
formin FH5 and regulates rice morphogenesis. Plant Cell 23: 681–700
Zhao ZY, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S,
Hondred D, Seltzer J, et al (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44: 789–798
Zhao Z-Y, Gu W, Cai T, Tagliani LA, Hondred D, Bond D, Schroeder S,
Rudert M, Pierce DA (2001) High-throughput genetic transformation
mediated by Agrobacterium tumefaciens in maize. Mol Breed 8: 323–333
Zhao ZY, Ranch J (2006) Transformation of maize via Agrobacterium tumefaciens using a binary co-integrate vector system. Methods Mol Biol
318: 315–323
Zheng J, Fu J, Gou M, Huai J, Liu Y, Jian M, Huang Q, Guo X, Dong Z,
Wang H, et al (2010) Genome-wide transcriptome analysis of two maize
inbred lines under drought stress. Plant Mol Biol 72: 407–421
Zhou S, Wang Y, Li W, Zhao Z, Ren Y, Wang Y, Gu S, Lin Q, Wang D,
Jiang L, et al (2011) Pollen semi-sterility1 encodes a kinesin-1-like protein important for male meiosis, anther dehiscence, and fertility in rice.
Plant Cell 23: 111–129
Zhou S, Wei F, Nguyen J, Bechner M, Potamousis K, Goldstein S, Pape L,
Mehan MR, Churas C, Pasternak S, et al (2009) A single molecule
scaffold for the maize genome. PLoS Genet 5: e1000711
Zou LP, Sun XH, Zhang ZG, Liu P, Wu JX, Tian CJ, Qiu JL, Lu TG (2011)
Leaf rolling controlled by the homeodomain leucine zipper class IV gene
Roc5 in rice. Plant Physiol 156: 1589–1602
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