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Plant Molecular Biology Reporter 22: 7–14, March 2004
© 2004 International Society for Plant Molecular Biology. Printed in Canada.
Commentary
Lethality of Inducible, Meristem-Localized Ectopic
␤-Glucuronidase Expression in Plants
FUSHI WEN1, HO-HYUNG WOO1,2, ANN M. HIRSCH2 and
MARTHA C. HAWES1,*
1
Department of Plant Sciences, Division of Plant Pathology and Microbiology,
University of Arizona, Tucson, AZ 85721; 2Department of Molecular, Cell and
Developmental Biology, University of California, Los Angeles, CA 90095-1606
Abstract. GUSA from Escherichia coli, encoded by the uidA gene, has been successfully
used as a plant reporter system for more than a decade with no reported deleterious effects.
However, when expressed in coordination with a UDP-glucuronosyltransferase isolated
from the root cap meristem of Pisum sativum (PsUGT1) at the onset of mitosis, GUSA expression was lethal in pea, alfalfa, and Arabidopsis thaliana. These unexpected results indicate that, under some circumstances, using GUSA in plants is incompatible with life and
suggest that the cell-specific lethal phenotype might be useful in selecting for genes specifically involved in regulating the G2-M phase of the cell cycle.
Key words: cell cycle, glucuronidase, lethality, meristem, reporter gene, root cap
Abbreviations: FCC, factor controlling cell cycle; GlcA, glucuronic acid; GUS, βglucuronidase; SL, saccharide lactone; UGT, UDP-glucuronyltransferase.
Lethality of
Introduction
ectopic
β-glucuronidase expression
Wen et al.
UDP-glucuronyltransferase (UGT) and β-glucuronidase (GUS) function together
to modulate the activity of steroid hormones and other regulatory molecules by
means of reversible conjugation (Nebert, 1991). In mammals, loss of function mutation in UGT or GUS is lethal, but a key role for these enzymes in higher plant
metabolism has only recently been proposed (Sasaki et al., 2000; Woo et al.,
1999, 2003). GUS forms the basis for an inexpensive quantitative assay because it
enzymatically cleaves glucuronic acid (GlcA) from a range of artificial substrates
whose levels can be measured by a simple colorimetric reaction (Levvy, 1952;
Lopez de Alda and Barcelo, 2001). The E. coli gene uidA encodes a heat-stable,
protease resistant exo-GUS with broad pH range and broad substrate specificity.
These properties have made it highly desirable as a plant reporter gene (Jefferson
et al., 1986; Jefferson, 1987). Although GUS activity is ubiquitous in plants, with
measurable activity in dozens of species and in most tissues, the endogenous enzyme exhibits a narrow pH optimum (<5.0) and a short half-life (Hansch et al.,
*
Author for correspondence. e-mail: [email protected]; fax: 1-520-621-9290;
ph: 1-520-621-5490.
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8
Wen et al.
1995; Hodal et al., 1992; Hu et al., 1990; Plegt and Bino, 1989; Schulz and
Weissenbock, 1988). Therefore, little or no background interference occurs in assays carried out under conditions optimal for GUSA. The E. coli gene has been
used routinely in thousands of experiments to define expression of hundreds of
plant genes, without reported negative effects on plant growth, development, or
cellular function. This includes more than 1000 field trials and several hundred
commercial releases of GUS-expressing crop species (Gilissen et al., 1998).
GUSA has also been successfully used as a reporter to detect environmental toxins in soil, air, and water (George et al., 2001; Kovalchuk et al., 2000, 2001a,
2001b; Legler et al., 2002; Wielbo and Skorupska, 2001). An absence of reported
toxicity has strongly suggested that ectopic GUSA expression in plants is benign
(e.g., Jefferson et al., 1987; Wilson et al., 1992).
In pea, expression of a meristem-localized gene encoding a UDPglucuronosyltransferase (PsUGT1) is correlated with the early stages of cell division in the root cap (Brigham et al., 1998; Woo and Hawes, 1997). PsUGT1 was
isolated by differential screening from the root cap meristem, in which mitosis
can be induced synchronously by the nondestructive removal of border cells from
the root cap periphery (Figure 1). Inhibition of enzyme activity by expressing
PsUGT1 antisense mRNA under the control of the PsUGT1 promoter in transgenic plants is lethal, and reduced or increased expression has a marked effect on
cell cycle duration, growth rate, and time required for completion of a life cycle
(Woo et al., 2003). These data argue that PsUGT1, which is expressed in all actively dividing tissues, plays a key metabolic role by modulating activity of a biologically potent structural or regulatory molecule. Preliminary results indicate that
PsUGT1 glycosylates a single aglycone with properties of a flavonoid (Woo et al.,
1999, 2002). Here we report that uidA, when expressed under the control of the
PsUGT1 promoter, is as toxic in alfalfa and pea as transformation with a known
cytotoxic gene.
Materials and Methods
Six independent experiments using 5 independently derived PsUGT1-uidA constructs to transform alfalfa were carried out according to established protocols
(Woo et al., 1999; 2003; 2004). Twelve independent experiments were performed
with Agrobacterium rhizogenes to establish transformed hairy root clones, using 5
independent PsUGT1-uidA constructs, according to established protocols (Woo et
al., 1999). Transformation was confirmed by means of Southern blot analysis, and
GUS activity was detected with histochemical staining (e.g., Jefferson, 1987; Jefferson et al., 1986, 1987).
Root cap turnover was induced by removing border cells (Figure 1), and
roots were treated for 20 min in water (0) or in 10-100 mM saccharide lactone
prior to incubation of seedlings overnight on damp filter paper.
Results and Discussion
Obtaining a single alfalfa plant expressing PsUGT1-GUSA was not possible
(Table 1). Successful transformation of plants with PsUGT1-GFP ruled out a
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Lethality of ectopic β-glucuronidase expression
9
Root cap
meristem
Border
cells
Figure 1. Dynamics of root cap development and PsUGT1 expression. A group of meristematic cells
[1], distinct from the root apical meristem, gives rise by mitosis to cells that differentiate
progressively into gravity sensing cells [2] and secretory cells [3] before separating from the cap
periphery as populations of root border cells [4] (Barlow, 1975). Mitosis in the cap meristem, and cap
turnover, is suppressed indefinitely (presumably at the G2 phase of the cell cycle) once a speciesspecific number of border cells accumulates on the cap periphery. When border cells are removed by
dipping the root tip into water or by wiping the cap with a tissue, mitosis in the cap meristem is
induced within 5 min, cap turnover resumes, and new border cells can be collected from the cap
periphery within 15 min. Mitosis returns to background levels within 1-2 h (Brigham et al., 1998;
Woo et al., 1999). Over a 24-h period, newly synthesized cells differentiate progressively until a
species-specific number of new border cells surrounds the cap periphery, at which point cap turnover
ceases. Because cells generated by mitosis in the cap meristem ultimately separate from the cap
periphery as border cells, the number of border cells present on the cap periphery after 24 h is a
direct reflection of the number of cells generated by mitosis in the meristem during the 1- to 2-h
period when cap turnover is induced (Clowes, 1980). PsUGT1 mRNA levels increase just before
mitosis increases and return to background levels as mitosis subsides.
toxic effect of the promoter, and lethality of transformants carrying PsUGT1-DT
confirmed that the promoter was active. Regeneration of control plants expressing
GUSA under control of the CaMV35S promoter was also successful. A few KmR
transformants expressing the diphtheria toxin chain A gene survived, but
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Wen et al.
Table 1. Lethality of GUSA when expressed in alfalfa.
Construct
No. of Km
Transformants Per
Leaf Disk
% of KmR
Transformants
Expressing
Inserted Gene
PsUGT1-GUS
PsUGT1-sGFP
PsUGT1-DT
pBI121
none
+++++
+/-*
+++++
NA
>95%
NA
>95
R
*A small number of explants was obtained, but death occurred in early stages of development.
Table 2. Lethality of GUSA expressed in pea hairy roots.
Construct
No. of Hairy
Roots Per
Stem
% of KmR
Hairy Roots
Per Stem
% of KmR Hairy
Roots Expressing
Inserted Gene
PsUGT1-GUS
PsUGT1-sGFP
PsUGT1-DT
pBI121
3-5
15-25
0
15-25
40-45%
40-45%
not applicable
40-45%
< 5%
> 90%
not applicable
> 90%
the growth and morphology of these plants were severely affected, and death
occurred early in development. Surviving transgenic lines failed to express detectable GUS activity, suggesting that their viability can be attributed to repression of
cellular activity by an unknown mechanism. Similar results were obtained when
PsUGT1-uidA was expressed in pea hairy roots (Table 2). A small percentage of
root clones expressed PsUGT1-uidA (as in Woo et al., 1999, Figure 2), but the
clones died within 7-10 d. In A. thaliana, of 400 explants transformed with the
PsUGT1-uidA gene, fewer than 0.1% expressed measurable GUS activity, and
these individuals were short-lived (data not shown) (Woo et al., 2003).
Thus, despite a long record of successful use as a reporter gene throughout
diverse plant tissues and organs, GUSA appears to be lethal when expressed during the same temporal and spatial window of PsUGT1 expression. This surprising
result can be explained by the following hypothetical model. In plants, as in other
higher eukaryotes (e.g., Nebert, 1991), endogenous GUS and UGT normally use
reversible glycosylation to regulate biological activity of a molecule whose cellular levels must be tightly controlled to prevent toxic effects. In the root cap,
PsUGT1 would be predicted to glycosylate a factor controlling cell cycle (FCC)
and thereby prevent its activity as a repressor of mitosis. Once a set number of
cells has been induced to transition from the G2 phase and enter into mitosis,
FCC is again activated when GUS removes GlcA, and mitosis is thereby suppressed. Blocking PsUGT1 activity is lethal because mitosis cannot be activated,
and reducing its activity slows the cell cycle and reduces the number of border
cells produced by the cap in a 24-h period by 99% (Woo et al., 1999). Conversely,
ectopic expression of uidA is lethal because resulting high levels of the target
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Lethality of ectopic β-glucuronidase expression
*
10,000-
Number of border cells per root tip
11
*
*
9,000*
8,000-
7,000-
6,000-
5,000-
*
4,0000
10
20
30
80
Concentration of saccharide lactone (mM)
Figure 2. Increased mitosis in response to treatment with saccharide lactone (SL), an inhibitor of
GUS activity. Root tips were incubated in SL for 20 min following removal of border cells and then
incubated at room temperature on water agar overlaid with filter paper. The number of new cells
produced after 24 h was measured by direct counts, as described (Brigham et al., 1998). Values
represent means and standard errors from at least 3 replicate samples from 10 seedlings, in 3
independent experiments.
aglycone would be predicted, according to this hypothesis, to permit unregulated
cell division. In mammals, a high level of GUS activity is a marker for unregulated cell division associated with most metastatic cancers (Huang and Oliff,
2001).
If this hypothesis were correct, inhibiting endogenous GUS activity during
the critical window of time when mitosis is induced in the root cap meristem
would foster increased mitosis. This prediction was tested by using saccharide
lactone (SL), a specific inhibitor of GUS activity (Levvy, 1952) (Figure 2). If endogenous GUS in the root cap normally functions to suppress mitosis by activating a repressor aglycone, inhibiting GUS in wild-type plants during the period
following removal of border cells should result in an increased number of new
border cells after 24 h. When root cap meristem was incubated in SL for 20 min
after border cell removal (i.e., when the G2-M switch normally occurs), a dosagedependent increase in the number of new border cells was observed (Figure 2).
Treatment with 100 mM or more resulted in seedling death (not shown). These
data support the hypothesis that PsUGT1 and GUS in the root cap operate together to reversibly glycosylate a molecule that directly or indirectly regulates the
cell cycle. The data may also help to explain why ectopic expression of GUSA is
lethal when expressed with PsUGT1.
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Wen et al.
Several reasons may exist to explain why the toxicity of ectopic GUSA has
not previously been reported. Expression of a wild-type phenotype in a transgenic
plant does not necessarily mean the inserted gene is benign (Hirschi, 2003).
Achieving an observable phenotype by altering any given gene product depends
on numerous factors, including concentration, timing, intracellular localization
and solubility, and tissue specificity. A failure of any one of these factors results
in the lack of an observable phenotype in the recovered plant. On the other hand,
lethality may have been overlooked. Regeneration of transformed plant lines constitutes a process of selection for individuals that have overcome lethal or deleterious effects. The lines most likely to be recovered are those that activate
compensatory mechanisms or fail to express the altered gene at times and places
where its effects are most likely to be damaging. Thus, in our experiments, regenerated lines of pea and A. thaliana transformed with PsUGT1-uidA were enriched
for individuals not expressing the transgene, with rare individuals exhibiting transient expression of the reporter gene. Without previous knowledge of toxicity in
pea hairy roots, in which lethality is a positive phenotype (Woo et al., 1999), the
lack of plants with stable reporter gene expression might have been dismissed or
misinterpreted. For example, alfalfa expressing uidA under the control of the
cyclin promoter CycD31, which like PsUGT1 is expressed at the onset of mitosis,
has been successfully regenerated. However, the transgene is not stably expressed
in the root meristem (Foucher and Kondorosi, 2000). These unexpected observations were interpreted as tissue-specific cyclin gene expression, but tissue-specific
toxicity is an alternative explanation. Lethality may only occur when GUSA is expressed in the root cap meristem. Although gene expression in the root cap is not
well characterized, this organ controls root movement and development, which in
turn modulate the architecture of the entire plant (Aiken and Smucker, 1996;
Jiang and Feldman, 2003; Tsugeki and Federoff, 1999). In recognition of these
properties, Darwin (1896) likened the root tip of plants to the central nervous system, which exerts a similar comprehensive regulatory effect in animal development.
The observation that GUSA has been expressed in plants under the control
of myriad promoters without reported problems may illustrate the principle of a
gun without a bullet and/or a bullet without a gun. However dangerous they may
be when together in a particular configuration in time and space, each is perfectly
harmless alone. These data highlight the value of systems like the inducible root
cap model in which gene expression can be evaluated in a controlled context on a
minute-by-minute basis (Hawes et al., 2003). Furthermore, they suggest that the
uidA gene–mediated lethality might be useful as a stage-specific reporter to select
for unknown genes that play a key role at the same phase of the cell cycle as
PsUGT1.
Acknowledgments
We acknowledge support from grants to M.C.H. and H.H.W. from the Department
of Energy, the US Department of Agriculture, the National Science Foundation,
and the College of Agriculture and Life Sciences at the University of Arizona.
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