The Plant Cell, Vol. 1, 403-413,April 1989 O 1989 American Society of Plant Physiologists
Alterations of Endogenous Cytokinins in Transgenic
Plants Using a Chimeric lsopentenyl Transferase Gene
June 1. Medford,” Roger Horgan,b Zaki El-Sawi,b and Harry J. Klee”,’
a
Plant Molecular Biology Group, Monsanto Company, 700 ChesterfieldVillage Parkway, St. Louis, Missouri 63198
The University College of Wales, Department of Botany and Microbiology, Aberystwyth SY23 3DA, United Kingdom
Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We
genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a
heat-inducible promoter (maize hsp70). The chimeric hsp7O isopentenyl transferase gene was transferredto tobacco
and Arabidopsis plants. Heat inductionof transgenic plants caused the isopentenyl transferase mRNA to accumulate
and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5’-monophosphate twofold. At
the control temperature zeatin riboside and zeatin riboside 5‘-monophosphate in transgenic plants accumulated to
levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various
aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area,
and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither
tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering.
Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the
uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in
cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent
cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shockedtransgenic
plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed
differentiation pattern were found with increased cytokinin levels, this process may be controlled by components
other than absolute cytokinin levels.
INTRODUCTION
In development, gene expression is coordinated in a temporal and spatial manner. Development in plants can be
described by two processes. One process, growth, involves change in cell size and number. A second process,
differentiation, encompasses modifications ranging from
subtle changes in cellular metabolism to physiological and
morphological modifications. The phytohormones appear
to play an important role in regulating plant growth and
differentiation. Evidence for this comes both from correlative studies, which attempt to relate endogenous hormone
concentrations to specific physiological processes, and
from effects of externally applied hormones. However,
both lines of evidence are open to criticism. Correlative
studies are frequently unable to distinguish between cause
and effect, and the results of externa1 applications of
hormones can only be interpreted correctly in relation to a
number of incompletely understood phenomena. These
’ To whom correspondenceshould be addressed.
phenomena include mechanisms of uptake, transport, inter- and intracellular localization, the ability or inability of
cells and tissues to respond to the phytohormone, and
metabolic pathways leading to activation/deactivation. An
approach to eliminating some of these problems is to
develop a technology for directing in vivo production of
phytohormones. This paper describes such an approach
for cytokinins.
Cytokinins are a class of phytohormones first defined as
substances that stimulate cell division in cultured tobacco
cells. However, externally applied cytokinins exhibit a wide
variety of effects. These include shoot initiation from callus
cultures, promotion of axillary bud growth, directed transport of nutrients, stimulation of pigment synthesis, inhibition of root growth, and delay of senescence. Given the
problems with exogenous application and pleiotropic effects, the possible role of endogenous cytokinins in plant
growth and differentiation is difficult to assess.
Compounding the previously mentioned problems is a
lack of knowledge concerning the regulation of cytokinin
biosynthesis. In terms of sites of biosynthesis, high cyto-
404
The Plant Cell
kinin levels have been reported to occur in roots, immature
leaves, and apical buds. However, demonstrating that
these tissues are actual biosynthetic sites is difficult since
the precise pathway of cytokinin production in plants has
not been rigorously established. In contrast, the pathway
for cytokinin biosynthesis directed by the plant pathogen,
Agrobacterium tumefaciens, is well established, The A.
tumefaciens cytokinin biosynthetic gene codes for the
enzyme isopentenyl transferase (ipt) (Akiyoshi et al., 1984;
Barry et al., 1984). This enzyme catalyzes the condensation of isopentenyl pyrophosphate and adenosine monophosphate giving isopentenyl AMP (iPMP) (McGaw, 1987).
In tumorous plant tissue this enzyme may represent the
rate-limitingstep in cytokinin biosynthesis (Akiyoshi et al.,
1983). Furthermore, this enzyme may have a role in nontumorous tissues since a partially purified enzyme with ipt
activity has been reported in tobacco cells (Chen and
Melitz, 1979).
Although details of cytokinin biosynthesis are not clear,
details relating to cytokinin metabolism and its role in
regulatingendogenous levels are somewhat clearer. There
is some evidence that the free base may be the most
biologically active form of cytokinin (Laloue and Pethe,
1982). Thus, most cytokinin metabolic processes lead to
a reduction in biological activity (McGaw et al., 1984).
However, the reversibility of some base/nucleoside/nucleotide modifications suggests that these may be endogenous mechanisms for regulating the leve1 of the biologically active cytokinins and thereby serve as a potential
way to fine tune any type of developmental regulation.
Some metabolic processes, such as side chain cleavage
and N-glycosylation, are irreversible and result in removal
of the cytokinins from the pool of biologically active material.
With the accessibility of cloned phytohormone biosynthetic genes from A. tumefaciens, endogenous cytokinin
levels can be manipulated. Constitutive production of cytokinins from fusion of the ipt gene to a constitutive promoter produced shooty plants that failed to root (Klee et
al., 1987). A system for regulatedcytokinin production was
designed to study better their role in plant development.
One type of regulatedexpression is that of the heat shock
genes. In response to elevated temperatures, organisms
terminate transcription and translation of most proteins
and initiate synthesis of a set of proteins known as the
heat shock (hsp) proteins (Bond and Schlesinger, 1987;
Key et al., 1987). Previously, Rochester et al. (1986) described the isolation of a maize hsp70 promoter, which
allowed thermal induction of a chimeric gene in transgenic
petunias. This promoter was fused to the A. tumefaciens
ipt gene and the chimeric gene (hs-ipt) introduced into
plants. We describe our experiments using thermal induction of the hs-ipt gene to examine which processes of
plant development are, and which processes are not,
affected by alterations of endogenous cytokinin levels.
RESULTS
Construction of a Heat-lnducible ipt Gene
hsp70 prometer, including
A 700-bp fragment of the Inaize
the entire 120-bp heat shock (hs) nontranslated
leader,
was fused to the ipt gene (Figure 1A). The nontranslated
leaderwas included
studies have suggested that there may be selected translation of heat shock
genes during heat shock (McGarry and Lindquist, 1985).
The chimeric gene was inserted into the binary plant
transformationvectors pMON505 and pMON850 to create
pMON595 and pMON601, respectively (Figure 1B). Transgenic tobacco and Arabidopsis plants were produced and
DNA gel blot hybridization confirmed that the regenerated
plants contained the hs-ipt gene (data not shown). Progeny
A.
Transcription
Stari
Bgl'l
BamHl
q , L d , - n - n - n 4 lsopentenyl Transferase I
tis
nontranslated
leader
B.
Balll
RB
pMON595
13469
pMON601
15766
RB
u
Figure 1. Construction of the Chimeric hs-ipt Gene.
(A) Structure of the chimeric gene. A 700-bp fragment containing
the maize hsp70 promoter and 5'-nontranslated leader sequence
was fused to the ipt coding region from pTiT37. This promoter
fragment contains two heat shock elements (HSE) located at
-106 and -54 bp from the transcription start site. The nontranslated leader consists of 120 bp derived from maize (\\\\\)
and 20 bp of linker 3' to the Bglll site.
(B) Plant transformation vectors, pMON595 and pMON601. Both
vectors contain ColEl-type (pBR) and broad host range (RK2)
origins allowing replication in Escherichiacoli and A. tumefaciens,
respectively. Each plasmid contains a 2.5-kb fragment of pTiT37
containing the T-DNA right border (RB) for integration into the
plant genome and a marker gene, nopaline synthase (NOS). The
spectinomycin resistance gene from Tn7 (Tn7 spc/str) allows
selection in bacteria. Plasmid pMON595 contains a neomycin
phosphotransferase gene (NOS/NPT/NOS) allowing kanamycin
selection in plants, whereas pMON601 contains a gentamicin
acetyltransferase AAC(3)-IV gene allowing gentamicin selection in
plants.
Alteration of Endogenous Cytokinins
of the transgenic plants were identified by germination
assays on media with antibiotics (kanamycin for tobacco
or gentamicin for Arabidopsis) or by nopaline synthase
assays (Otten and Schilperoort, 1978).
A.
405
B.
Heat Induction of ipt Gene Expression
Based on data from preliminary experiments (Medford,
Winter, and Klee, 1988), temperatures of 42°C (Arabidopsis) and 45°C (tobacco) were chosen. Arabidopsis and
tobacco plants were heat shocked in vivo for 2 hr, and
immediately afterward, the hs-ipt mRNA could be readily
detected (Figure 2). In the uninduced transgenic tobacco
plants, the ipt message could not be detected, even with
20 jug of poly A+ RNA per lane and extensive exposure of
autoradiograms (Figure 2B). This indicates that the levels
of uninduced ipt mRNA in transgenic plants must be less
than 1.0% of the induced levels.
Heat Induction of Cytokinins in Transgenic Plants
The transgenic plants were examined for heat-induced
accumulation of Cytokinins. Plants were heat shocked and
allowed to recover for 4 hr, and tissue was harvested,
frozen in liquid nitrogen, and lyophilized. The major cytokinins previously identified in tobacco crown gall tissue
(containing the ipt gene) were measured using the 2H/15N
technique of Scott and Morgan (1984). iPMP and the
cytokinin nucleotide, ZMP (ribosyl zeatin 5'-monophosphate) were measured by selective ion monitoring using
2
H internal standards whereas Z (zeatin, the free base),
ZR (ribosyl zeatin), and Z7G (zeatin 7-0-o-glucoside) were
measured using 15N internal standards. No precautions
were taken to minimize phosphatase action during extractions since the method used can account for this. However,
recovery of the 2H-iPMP internal standard was generally
very low, suggesting that the values for the level of this
compound are probably underestimates.
The level of all Cytokinins measured was considerably
elevated in heat-shocked transgenic plants (Table 1). The
level of Z increased 52-fold, ZR increased 23-fold, and
ZMP increased twofold. In addition, the presence of a
considerable quantity of Z7G in the heat-shocked plants
indicates that a significant amount of metabolic inactivation
of cytokinins occurred in the 4 hr following heat shock.
Relative to cytokinin levels found in tobacco crown gall
tissues, the base (Z) and riboside (ZR) are much higher in
the transgenic heat-shocked plants. The most likely reason
for this is that the production of cytokinins in the transgenic
plants takes the form of a rapid biosynthesis pulse induced
by the heat shock, whereas in crown gall tissues, cytokinins are produced by a slower continuous process.
870 bp
870 bp
Figure 2. Analysis of ipt mRNA Expression in Transgenic Plants.
Transgenic plants were heat-shocked at 42°C (Arabidopsis) or
45°C (tobacco) for 2 hr. RNA was isolated immediately after heat
shock.
(A) 40 M9 of total RNA isolated from transgenic Arabidopsis plants
at the control and heat-shocked temperature. The arrow indicates
the 870-bp chimeric ipt mRNA.
(B) Twenty micrograms of poly A+ RNA isolated from transgenic
tobacco plants at the control and heat-shocked temperature.
It is of interest to note that, in transgenic plants maintained at the control temperature, the levels of ZR and
ZMP are elevated (threefold and sevenfold, respectively).
This is indicative of a slight "leakiness" of the gene at the
control temperature. Although this was not observable at
the mRNA level, a small increase in cytokinin production
integrated over a period of time could result in the increased levels of ZR and ZMP (Table 1).
Effects of Cytokinin Alterations on Growth and
Differentiation
Changes in the Shoot System of Tobacco
Transgenic tobacco plants were examined for effects due
to altered cytokinin levels. Even without thermal induction,
tobacco plants containing the hs-ipt gene showed a reduction in height (Table 2). However, the number of nodes in
wild-type and transgenic plants was not significantly different. Thus, whether the height reduction is a result of
decreased number of nodes, internode length, or a combination thereof, could not be clearly determined. In the
transgenic plants there was a reduction in xylem content.
Mature xylem in transgenic plants represented only 70%
406
The Plant Cell
Table 1. Cytokinin Levels in Wild-Type and Transgenic Tobacco Plants
Cytokinin Level, ng/g
Wild-type
Wild-type, heat-shocked
Transgenic
Transgenic, heat-shocked
ND
iPMP
Z
ZR
ZMP
Z7G
14
18
31
102
46
18
23
1450
44
48
94
2230
86
72
640
1667
ND"
ND
ND
700
Plants were heat shocked for 2 hr. Four hours afterwards, cytokinins were extracted from leaf tissues and quantitated as described in
Methods.
a ND, not determined.
of the controls. In a similar pattern, leaf size in the transgenic plants was reduced, representing only 73% of the
controls.
Heat inductions were given at weekly intervals, starting
when the plants had four true leaves greater than 3 cm in
length. At this point the plants have formed only 1O to 12
of their 30 to 40 nodes. Even in the absence of heat
induction, transgenic plants again showed a reduction in
height (Figure 3). In response to heat induction, wild-type
plants showed no changes in height or other morphological
factors. Unexpectedly, heat induction of cytokinins in the
transgenic plants producedno changes beyond those seen
in the uninduced state (Figure 3). These results, with
effects in uninduced plants and no further effects in induced plants, were observed in several independent transgenic plants.
Cytokinins have been thought to enhance the onset of
flowering in several species (Bernier, 1988). In the transgenic tobacco plants, despite the increased leve1 of cytokinins from uninduced or induced production, there was
no significant change in the days to flowering (Table 2). In
addition to flowering, the enhanced production of cytokinins in uninduced transgenic plants did not perturb development of fertilized ovules into seeds. In heat-shocked
plants, both wild-type and transgenic, the elevated temperature itself (45OC) caused ovule abortion.
Two other aspects of cytokinin alterationson the pattern
of plant growth were noted. First, axillary bud growth in
the transgenic plants was significantly greater than that of
wild-type plants (Figure 4, A and B). Although the greatest
differences were found at the uppermost nodes, this difference was true for all nodes of the plant (Figure 4C). A
second aspect of plant growth, heterostylic development
(which was previously reported in grafted shoots containing the ipt gene [Wullems et al., 1981]), was notably absent
in the uninduced transgenic plants. After heat induction,
heterostylic development was found, but since this was
true for both wild-type and transgenic plants, it was impossible to distinguish between the effects from heat
shock and effects from elevated cytokinins.
Changes in the Shoot System in Arabidopsis
Heat induction of cytokinins in Arabidopsis started with
plants 7 days after sowing (16-hr photoperiod) and at 3day intervals thereafter, up to the point of anthesis. As in
tobacco, the thermal induction of cytokinins in transgenic
Arabidopsis plants produced no further changes in the
development of the shoot system. Thermal induction of
cytokinins was performed throughout floral development.
No differences in floral differentiation that could be attributed to cytokinin overproduction alone were observed. As
in tobacco, there was no effect of uninduced cytokinins on
ovule development, and heat induction at 42OC produced
abortion in both the transgenic and control plants.
Arabidopsis, a facultative long day plant, grown under
long days (16 hr) will go from seed to flowering in 6 weeks.
Transgenic plants grown under these conditions showed
no discernible reduction in xylem proliferation as seen in
transgenic tobacco plants. However, under short day (8
hr) conditions, Arabidopsis plants required 5.5 months to
go from seedling to flowering stage. When floral stem
cross sections of short day plants were examined, the
transgenic Arabidopsis plants had reduced xylem proliferation similar to that found in the transgenic tobacco plants
(Figure 5).
Table 2. Comparison of Wild-Type and Transgenic Tobacco
Plants
Height, cm
Node number
Stem area
Days to flowering
Transgenic
Wild-Type
80.3 f 5.9
35.1 -C 1.7
3.0 C 0.7
71.9 f 2.6
94.7 f 12.0
36.9 f 2.2
4.1 f 0.8
72.6 f 4.9
Measurements were done on F, progeny from a cross of a
transgenic plant to a wild-type plant. Transgenic progeny were
identified by nopaline synthase assays. Details of measurements
are found in Methods.
Alteration of EndogenousCytokinins
80
407
made conclusions difficult. Plants grown in short days were
not examined for axillary bud growth.
Changes in the Root System of Transgenic Plants
h
60
v
CI
r
w
.-
Q,
I
o
*-
c 40
a
ul
v)
c
E
I-
20
O
O
2
4
6
Number of Heat Shocks
In transgenic tobacco and Arabidopsis plants, the primary
root elongated at a slower rate than that of wild-type
plants (70% for tobacco, 68% for Arabidopsis). In both
species lateral roots were initiated in a pattern indistinguishable between transgenic and wild-type plants. However, the root tip region of transgenic plants (Figure 6, A
and B) was wider and shorter than that of wild-type plants.
In addition, root hairs in transgenic plants emerged closer
to the tip. This suggests that the zone of elongation is
reduced in transgenic plants. In wild-type plants, longitudinal files of maturing rectangular cells could be easily
traced. However, in the transgenic plants, the periclinal
walls of the maturing cells were often extended, causing
a disruption of the orderly pattern of growth (Figure 6A).
This pattern of growth was not readily discernible in the
young seedling roots, but became more obvious as the
roots aged. At maturity, this effect resulted in a reduction
of root mass (Figure 7).
DISCUSSION
Figure 3. Reduction of Height in Tobacco Plants Containing the
hs-ipt Construct.
Heights of individual plants were measured at the first anthesis.
Bars indicate standard deviation. Each point consists of data from
at least six plants.
In Arabidopsis, exogenous application of cytokinins has
been reported to enhance the onset of flowering (Michniewicz and Kamienska, 1965; Besnard-Wilbaut, 1981). To
test the effect of enhanced cytokinin levels on flowering,
plants were grown in short (8-hr) days. Two and a half
months after sowing, one set (wild-type and transgenic)
was given heat shocks every 3rd day for a period of 3
weeks. Heat shocks were conducted at times so as not
to disrupt the photoperiod. Two months after the conclusion of the heat induction (5.5 months from the start of the
experiment), the plants flowered. However, there was no
significant difference in the time of flowering between
plants containing the hs-ipt gene and wild-type plants. This
was true for both those plants exposed to thermal induction and those maintained at the control temperature.
An additional effect of cytokinin overproduction in nonheat-shocked tobacco plants was the enhanced development of axillary buds. Axillary growth in transgenic Arabidopsis plants grown in long days was somewhat enhanced
in the rosette nodes, although the extremely small size
Cytokinins are believed to play regulatory roles in two
processes that describe plant development: growth and
differentiation (Finkelstein et al., 1987; King, 1988). Elucidation of processes that cytokinins affect has come, in
part, from studies where the hormone is applied externally.
This approach presents added complexities that limit definitive conclusions. This is problematic since it is equally
important to understand the processes that are not effected by phytohormones as well as those that are
effected.
To regulate endogenous cytokinin levels, we fused an
ipt gene from A. tumefaciens to a maize hsp7O promoter.
After heat induction, the ipt mRNA was readily detectable
in transformedArabidopsis and tobacco plants (Figure 2).
Although the mRNA could not be detected without heat
induction,the threefold and sevenfold increase in the levels
of ZR and ZMP (respectively) in the uninduced transgenic
plants indicates that the chimeric gene must be expressed
to some extent at control temperatures. Although expression of the maize hsp70 promoter at control temperatures
must be nominal, even a low leve1 of constitutive ipt gene
expression at control temperatures could account for the
accumulation of cytokinins, since expression of ipt mRNA
from the promoter normally found in the A. tumefaciens Ti
plasmid (a relatively weak promoter) is enough to alter
plant morphology (Akiyoshi et al., 1983).
408
The Plant Cell
9
12
15
18
21
NODE NUMBER (from apex)
Figure 4. Comparison of Growth of Axillary Buds in Wild-Type and Transgenic Tobacco Plants.
(A) and (B) Growth of axillary bud at 12th node (from shoot apex) in wild-type (top) and transgenic (bottom) plants.
(C) Fresh weight (mg) of axillary buds from nodes of wild-type and transgenic plants.
Presumably, the uninduced expression from the maize
hsp70 gene occurs equally throughout the plant. To investigate tissue distribution of the heat shock response, the
hsp70 promoter was fused to the reporter gene, 0-glucuronidase (GUS). At the control temperature, no detectable
GUS activity was found in transgenic tobacco and Arabidopsis plants containing the hs-GUS fusion. However, 4
hr after heat induction, histochemical analysis showed
GUS expression in all major organs and tissues (data not
shown).
Accumulation of cytokinins (Table 1) demonstrates that
the chimeric hs-ipt gene is producing a functional enzyme,
and that the plant is converting iPMP into the cytokinins,
Z, ZR, and ZMP. Despite large increases in cytokinin levels
following heat induction, no further effects beyond those
seen at the control temperature were observed. This was
unexpected since our similar experiments with the same
promoter and auxin biosynthetic genes showed dramatic
alterations in phenotype after heat induction (Medford and
Klee, 1988). There are several possible explanations, none
Alteration of Endogenous Cytokinins
409
with xylem reduction in both transgenic tobacco plants
and transgenic Arabidopsis plants grown in short days. In
Arabidopsis plants grown in long days, this effect was less
pronounced. Since Arabidopsis plants mature rapidly under long day conditions, a small reduction in xylem proliferation would not be discernible. However, growth of
Arabidopsis plants under short days takes 5 to 6 months
and would allow small changes in xylem formation to
become more apparent.
In addition to changes in the amount of xylem, axillary
bud growth in the transgenic tobacco and Arabidopsis
plants was increased. This agrees with exogenous application studies where cytokinins were applied directly to
the axillary buds (Tamas, 1987). Our previous studies on
auxin overexpression in transgenic petunias (Klee et al.,
1987; Medford and Klee, 1988) suggested that auxin
suppression of axillary buds is a result of auxin levels
rather than gradients. Our work here indicates that increased cytokinin levels enhanced the growth of the axil-
Figure 5. Cross-section of Wild-Type and Transgenic Arabidopsis
Floral Stems Grown in Short Days.
Wild-type stems (top) and transgenic stems (bottom) stained with
toluidine blue (magnification x50).
of which are mutually exclusive. First, since the increased
cytokinin level is most likely transient, it is possible that
the altered levels of active cytokinins do not persist long
enough for a phenotypic response to be observed. A
second possibility is that there may be a direct or indirect
induction of negating factors such as metabolic enzymes
like cytokinin oxidase. The high level of Z7G 4 hr after heat
shock supports an enhancement of cytokinin metabolism.
A third possibility is that there is a lack of corresponding
level of cellular factors for perception of the high level of
cytokinins.
In uninduced transgenic plants, the elevated cytokinin
levels altered some aspects of plant development. In the
transgenic plants, there was a reduction in height, leaf
area, and stem width. The reduced stem width correlated
Figure 6. Effect of Increased Cytokinin Levels on Root Growth.
(A) Arabidopsis primary root at 14 days. Right, wild-type; left,
transgenic.
(B) Tobacco primary root at 12 days. Left, wild-type; right, transgenic (magnification of both figures is x50).
410
The Plant Cell
Figure 7. Cumulative Effects of Cytokinin Alterations on Tobacco
Root Growth at Maturity.
Left, wild-type; right, transgenic.
lary buds. Collectively, these data indicate that axillary bud
release is dependent on the auxin/cytokinin ratio within
the bud.
In both tobacco and Arabidopsis, the increase in cytokinins (both uninduced and induced) had no effect on flowering. We are unaware of any direct report of the effect of
cytokinins on flowering in tobacco. However, in Arabidopsis, two independent studies have shown that application
of exogenous cytokinin (kinetin) to the shoot apex resulted
in enhanced floral development (Michniewicz and Kamienska, 1965; Besnard-Wilbaut, 1981). The work of BesnardWilbaut (1981) showed that, for kinetin to be effective at
floral induction, the plants first had to reach a certain
developmental stage. In our studies, the elevated level of
cytokinins under non-heat-shocked conditions is presumably constant throughout development. In addition, heatinduced elevations of cytokinins were done when the
plants should have been competent to respond to the
cytokinin increase. The major difference between our study
and previous ones is that the endogenous production
presumably occurs throughout the entire plant, whereas
the exogenous cytokinins were applied directly to the
shoot apex. Thus, enhancement of flowering in Arabidopsis by exogenous cytokinins could be more a result of
selective nutrient redistribution to a competent apex rather
than a specific action of cytokinin per se. Alternatively, it
is possible that, in the transgenic Arabidopsis plants, there
was only a partial evocation, which would require extensive
analysis of the shoot tip to distinguish.
The effect of altered cytokinins on the root system was
similar in both tobacco and Arabidopsis. Root growth,
measured in terms of elongation, was reduced in the
transgenic plants. The reduction in the rate of root growth
corresponded to a decrease in the size of the zone of
elongation. A previous study has shown a correlation
between reduced size of the zone of elongation and reduced root growth rate (Hinchee, 1981). Another effect of
altered cytokinin levels on root growth in transgenic plants
was a disruption of the orderly pattern of longitudinal files
of cells. The similar phenomenon was observed in vitro
when kinetin was supplied exogenously to maize roots
and was attributed to dissolution of the middle lamella of
cell walls parallel to the root surface (Kappler and Kristen,
1986). In the transgenic plants, we noted that these walls
were often bulging, consistent with the exogenous application study. In the transgenic plants this effect became
more conspicuous as the roots grew. Since disruption of
cell organization was observed only in the roots, this
suggests that the root system may be more sensitive to
alterations in cytokinin levels than the shoot system.
Cytokinins have been described as compounds that, in
combination with auxin, delineate a differentiation pathway
(Wareing and Phillips, 1981). We noted that the major
developmental effects of altered cytokinin levels in the
transgenic plants were on aspects of plant growth and not
differentiation. Since these changes were found in a number of tissues (e.g., leaves, stems, roots), the mechanism
by which cytokinins bring about growth modifications may
be a fundamental change in aspects of cellular metabolism.
In altering the endogenous cytokinin levels in the transgenic plants, no alterations in the differentiation processes
were detected. We could not find any evidence for changes
in the plant's programmed pattern of differentiation. This
was true in both the shoot system and the root system
from two different plant families. The fact that the altered
endogenous cytokinin levels per se did not affect differentiation suggests that this process, unlike the process of
plant growth, may be controlled by components other than
absolute cytokinin levels.
METHODS
Vector Construction
A 700-bp fragment from the maize hsp70 promoter (Rochester et
al., 1986) was cloned into pUC119 for site-directed mutagenesis
using the procedure described by Kunkel (1985). A Bglll site was
introduced at the translational start site. This allowed the entire
promoter and 5'-nontranslated leader to be excised as a BamHIBglll fragment. This fragment was then cloned into the unique
Bglll site of pMON505 (Horsch and Klee, 1986), creating
pMON591. The ipt gene from pTiT37 (Goldberg et al., 1984) was
cloned into pMON591 as a 1.2-kb Bglll-EcoRI fragment, creating
pMON595 (Figure 1). The same fragments were inserted into the
plant transformation vector pMON850, creating the plasmid
pMON601 (Figure 1). For construction of the /i-glucuronidase
gene (GUS) fusion, the mutagenized heat shock promoter was
fused to a 1.9-kb Hindlll fragment from pMON9749 (Hinchee et
al., 1988) containing the entire GUS opening reading frame fused
to the nopaline synthase 3' sequence.
Alteration of Endogenous Cytokinins
Plant Materials
Nicotiana tabacum cv Samsun and Arabidopsis thaliana ecotypes
Columbia and RLD were grown in controlled environmentalchambers at 25OC, 50% relative humidity, and 16-hr day length. Tobacco plants received water and nutrients twice a day from
automatic watering systems, whereas Arabidopsis plants were
watered by hand and fertilized once a week. For one set of
experiments with tobacco, plants were grown in the winter months
in a greenhouse maintained at 25'C with supplemental lighting.
Greenhouse-grown plants were watered daily by hand and received a fertilizer once a week. For experiments on flowering in
Arabidopsis, plants were grown in a Percival set for short days (8
hr) with watering and fertilization by hand when required.
41 1
Heat-shocked tissue was placed in the substrate solution 4 hr
after return to the control temperature.
Thermal lnductions
Heat shocks were performed in vivo by temperature increases in
a controlled environmental chamber. To use the heat shock
promoter to create inducible changes in endogenous phytohormone levels, conditions were first defined that allowed both gene
induction in vivo and plant survival. Plants were heat-shocked by
a linear elevation of temperature over a 15-min period. The
elevated temperature was maintainedfor 2 hr with relative humidity of 70%, followed by a 15-min linear decline. The plants (both
Arabidopsis and tobacco) survived the heat shock at all temperatures tested.
Plant Transformation
RNA Gel Blot Analysis
Tobacco plants transformed with pMON595 were obtained using
the leaf disc method (Horsch et al., 1985). Arabidopsis plants
transformed with pMON601 were obtained using the method of
Lloyd et al. (1986) with modifications for gentamicin selection
(Hayford et al., 1988). Progeny of primary transformants were
identifiedby germinating seed on 1O0 pg/mL kanamycin (tobacco)
or 80 pg/mL gentamicin (Arabidopsis).
Growth Measurements
For measurements of root growth, sterilized seeds were germinated on agar medium (Murashige and Skoog, 1962) containing
30 g/L sucrose and Murashige and Skoog (MS) salts mixture
(GIBCO) at one-half the normal concentration. Petri dishes were
placed vertically and the length of the primary root measured after
5 days for Arabidopsis. For tobacco, the primary root was marked
5 days after germination and the distance from the initial mark to
the root tip measured at 12 days. Growth at the root tip was
observed directly or after brief staining in toluidine blue.
Tobacco leaf areas was measured as a product of the lamina
length and its width at the widest part (6 cm from the base). Stem
area was determined by tracing the stem cross-sections at node
14, followed by weight measurementsof the tracing. Stems were
sectioned by hand, stained with toluidine blue, and examined
under a microscope. Measurements of xylem proliferation were
done at nodes 7 and 14 on 12 separate plants. The extent of
xylem proliferation was determined by counting the mature elements in an individual file of cells. Height and cross-sectional area
determinations were done on a populationof transgenic and wildtype plants. Stem cross-sectional area was calculated by tracing
the stem diameter at the 15th node (from the base) on paper
followed by weighing the paper. Results were analyzed using the
Student's t test and were significant to P > 0.995.
GUS Histochemical Assays
Histochemical assays were performed using X-gluc (5-bromo-4chloro-3-indolyl-p-~-glucuronicacid, cyclohexylammonium salt,
ResearchOrganics)as a substrate (Jeffersonet al., 1987). Tissues
were sectioned by hand, placed into the substrate solution, vacuum infiltrated, and incubated at room temperature overnight.
RNA was isolated from plant tissue using precipitation with LiCl
(Rochester et al., 1986). Poly A+ RNA was prepared using
oligo(dT) column chromatography as described (Maniatis et al.,
1982). RNA was separated on 1.4% agarose gels with formaldehyde and transferred to a nylon membrane (Thomas, 1983).
Expression of ipt mRNA was analyzed by hybridization of the
cloned ipt gene to the RNA gel blots. Hybridizations were done
at 42°C for 16 hr in 50% formamide, 4 x SSPE, 4 x Denhardt's
solution, 1% SDS, and tRNA at 100 pg/mL (Maniatis et al., 1982).
The filters were washed twice for 20 min at room temperature in
0.5 x SSPE, 0.1% SDS followed by one wash at 65OC in the
same solution.
Cytokinin Analysis
Plants were heat-shocked as described above and 4 hr afterward,
leaf tissue (tobacco) or the entire rosette (Arabidopsis) was excised, frozen immediately in liquid nitrogen, and lyophilized. The
lyophilized tissues were extracted in 80% methanol with the
addition of 'H2-ZMP and iPMP, and '5N4-Z,ZR, and Z7G as the
interna1standards. The cytokinins were purified by reverse-phase
HPLC and measured by GC-MS-selective ion monitoring as the
permethyl derivatives, with the exception of Z7G, which was
analyzed as the trimethylsilyl derivative. The method used was
essentially that of Scott and Horgan (1984).
ACKNOWLEDGMENTS
We thank Jill Winter and Maud Hinchee for their helpful discussions. We thank Nancy Hoffmann for producing the transformed
tobacco plants and Yvonne Muskopf for assistance with transgenic Arabidopsis plants. We are also grateful to Steve Rogers,
Rob Horsch, and Robb Fraley who provide continuous support to
all our efforts.
Received February 6, 1989.
41 2
The Plant Cell
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DOI 10.1105/tpc.1.4.403
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