Plant Physiol. (1998) 116: 969-977
Controlled Cytokinin Production in Transgenic
Tobacco Using a
Copper-Inducible Promoter
Marian Jane McKenzie1, *,
Vadim Mett,
Paul Hugh Stewart Reynolds, and
Paula Elizabeth Jameson
Botany Department, University of Otago, Private Bag 56, Dunedin,
New Zealand (M.J.M.); Plant Improvement Division, The Horticulture
and Food Research Institute of New Zealand, Private Bag 11030, Palmerston North, New Zealand (V.M., P.H.S.R.); and Department of Plant
Biology and Biotechnology2, Massey University, Private
Bag 11222, Palmerston North, New Zealand (P.E.J.)
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ABSTRACT |
The
cytokinin group of plant hormones regulates aspects of plant growth and
development, including the release of lateral buds from apical
dominance and the delay of senescence. In this work the native promoter
of a cytokinin synthase gene (ipt) was removed and
replaced with a Cu-controllable promoter. Tobacco (Nicotiana
tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically
identical to controls under noninductive conditions in almost all lines
produced. However, three lines grew in an altered state, which is
indicative of cytokinin overproduction and was confirmed by a full
cytokinin analysis of one of these lines. The in vitro treatment of
morphologically normal Cu-ipt transformants with
Cu2+ resulted in delayed leaf senescence and an increase in
cytokinin concentration in the one line analyzed. In vivo, inductive
conditions resulted in a significant release of lateral buds from
apical dominance. The morphological changes seen during these
experiments may reflect the spatial aspect of control exerted by this
gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in
tobacco transformants can be temporally and spatially controlled by the
induction of ipt gene expression through the
Cu-controllable gene-expression system.
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INTRODUCTION |
The discovery of the plant hormone group the cytokinins and their
involvement in aspects of plant growth and development, such as cell
division (Skoog and Miller, 1957
), delayed senescence (Richmond and
Lang, 1957), and the release of lateral buds from apical dominance
(Sachs and Thimann, 1964), has led to the attempted manipulation of
these processes by altering the endogenous cytokinin content in plant
tissues. Since the major gene(s) involved in cytokinin production in
plants has not yet been isolated, a number of groups have utilized the
ipt gene from the plant pathogenic bacterium
Agrobacterium tumefaciens. This gene encodes the enzyme isopentenyl transferase, which catalyzes the rate-limiting step of the
cytokinin biosynthetic pathway (Akiyoshi et al., 1984; Barry et al.,
1984), in which 
2-isopentenyl PPi is condensed
with AMP to form isopentenyl AMP. The introduction of the
ipt gene into the plant genome results in elevated levels of
cytokinin in the transformed tissue (Smigocki and Owens, 1988;
Beinsberger et al., 1991; Yusibov et al., 1991; McKenzie et al., 1994),
together with associated morphological changes.
Plants transformed with highly expressing ipt genes produce
shoots that do not elongate, show a severe lack of apical dominance, have small, rounded leaves, and are not able to form roots
(Schmülling et al., 1989; Smigocki, 1991; Hewelt et al., 1994).
Whole plants transformed with ipt genes with weaker or
controlled expression also display the effects of cytokinin
overproduction, the most consistent of these being the release of
lateral bud growth from apical dominance (Medford et al., 1989; Smart
et al., 1991; Smigocki, 1991; Van Loven et al., 1993; Hewelt et al.,
1994; Faiss et al., 1997). This aspect of development is also inducible
by exogenous cytokinin application (Sachs and Thimann, 1964).
Additionally, a decrease in root production by ipt
transformants has been repeatedly observed (Medford et al., 1989;
Smigocki, 1991; Van Loven et al., 1993), and the leaves of
ipt transformants exhibit delayed senescence (Smart et al.,
1991; Li et al., 1992; Hewelt et al., 1994; Gan and Amasino, 1995;
Faiss et al., 1997). Delayed leaf senescence has also been observed
following the exogenous application of cytokinin (Richmond and Lang,
1957). Other reported effects of endogenous cytokinin overproduction
include the production of the defense-related genes for extensin,
chitinase, and PR1 (Memelink et al., 1987), and increased tuber
formation from potato plants (Ooms and Lenton, 1985).
The altered morphology observed in plant tissues transformed with the
native ipt gene has been clearly associated with a marked increase in cytokinin content (Budar et al., 1986; Smigocki and Owens,
1988; Beinsberger et al., 1991; Yusibov et al., 1991; McKenzie et al.,
1994). Additional work has been carried out using the CaMV 35S promoter
to drive ipt gene expression (Smigocki and Owens, 1988,
1989). This showed that the CaMV 35S promoter increased ipt
expression over that of the native promoter. However, the constitutive
expression of the ipt gene, through either its native promoter or the CaMV 35S promoter, prevented normal plant development, thereby precluding the study of cytokinin overexpression within the
whole plant.
To study the effects of cytokinin overproduction on normal plant
tissue, a number of groups have linked regulatable promoters to the
ipt gene. The most commonly used promoters have been those regulated by heat shock, and several groups have obtained whole plants
using such promoters to control ipt gene transcription (Medford et al., 1989; Schmülling et al., 1989; Smart et al., 1991; Smigocki, 1991; Van Loven et al., 1993). These plants were able
to form normal roots but were often smaller and displayed a greater
degree of axillary bud growth than control plants under both inductive
and noninductive conditions. Hormone analyses indicated that even under
non-heat-shock conditions transformed plants often contained higher
levels of cytokinin than did control plants. When heat shock was
carried out, cytokinin levels increased further, but this was not
necessarily accompanied by further morphological changes (Medford et
al., 1989; Smigocki, 1991). Thus, it seems that the heat-shock
promoters allow sufficient expression from the ipt gene
under noninductive conditions to alter plant morphology. Moreover, heat
shock itself may affect plant growth, and plant treatment prior to heat
shock may induce gene expression (Van Loven et al., 1993).
In moving away from the use of heat-shock promoters, a number of groups
have utilized promoters that allow temporal or spatial gene expression.
These have included promoters controlled by the external environment,
e.g. light (Beinsberger et al., 1991), wounding (Smigocki et al.,
1993), tetracycline (Redig et al., 1996; Faiss et al., 1997), and those
related to a particular tissue or developmental state, such as
fruit-specific (Martineau et al., 1994), hormone-specific (Li et al.,
1992), or senescence-specific (Gan and Amasino, 1995) promoters.
Generally, a higher level of control over ipt gene expression has been gained with these promoters than has been provided
by the heat-shock promoters. However, in many cases cytokinin production still appears to be dependent on treatment type as well as
on tissue type. In fact, Faiss et al. (1997) suggested that the
cytokinins are active only in the tissue in which they are synthesized.
Their conclusion is derived in part from grafting experiments in which
they showed that there is no influence over apical dominance or
senescence in wild-type tissue grafted onto transgenic rootstock. The
use of a tightly controlled, inducible promoter activated specifically
in the roots would not only avoid the possible compounding effect of
the graft union but would also pinpoint the key areas of cytokinin
control.
In the work reported here we controlled the expression of the
ipt gene in tobacco (Nicotiana tabacum L. cv
tabacum) using the Cu-inducible gene expression system (Mett et al.,
1993, 1996). This system is activated directly by Cu, allowing the
defined expression of genes to which it is linked, and has been shown to provide tight control over the expression of a GUS reporter gene,
such that GUS expression occurred only from the root tissue of tobacco
transformants in the presence of 50 µm
CuSO4 (Mett et al., 1996). This method of control
was considered ideal for supplying additional cytokinin to the plant,
since increased cytokinin production in the roots might enhance the
cytokinin supplied naturally to the other plant organs via the xylem.
The provision of excess cytokinin in the appropriate physiological
context could be expected to provide a particularly useful model
system. When this system was used, ipt gene expression
occurred in tobacco transformants in the presence of
Cu2+ but did not occur in its absence. The
controlled expression of the ipt gene resulted in increased
cytokinin levels, the breaking of apical dominance, and delayed leaf
senescence.
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MATERIALS AND METHODS |
PCR
Two primers were designed that flanked the coding and termination
regions of a previously isolated ipt gene sequence (McKenzie et al., 1994). PCR was performed using a Perkin Elmer-Cetus kit according to the manufacturer's instructions.
Vector Construction
The vector pMMACEipt was constructed in three steps: (a) The
promoterless ipt gene was cloned into the
EcoRI/XbaI sites of the plasmid pUC119/4MT3 (Mett
et al., 1996) following the four copies of the metal responsive
element. (b) A second NotI site was introduced at the end of
the ipt termination sequence allowing the NotI
fragment (containing the metal-responsive elements and the promoterless
ipt gene) to be cloned into the pACE-in-ART
vector (Mett et al., 1996). (c) The NotI fragment was cloned
into the NotI site of the binary vector pACE-in-ART,
creating pMMACEipt. A similar vector, pGA643/ACE1.6/4MT-40GUS (Mett
et al., 1996), was used for control transformations. This vector
contained the GUS gene instead of the ipt gene.
Southern Analysis
Genomic DNA was prepared from the leaves of young,
tissue-culture-grown tobacco (Nicotiana tabacum L. cv
tabacum) using a cetyltrimethylammonium bromide method based on that
described by Akama et al. (1992). The DNA was digested overnight with
EcoRI, subjected to gel electrophoresis, and blotted to a
Zeta Probe membrane (Bio-Rad) by capillary transfer (Southern, 1975).
The membrane was probed with the promoterless ipt gene,
which was labeled with [32P]dCTP (3000 Ci
mmol
1).
Plant Transformation
Leaf discs of tobacco were transformed by co-cultivation with
Agrobacterium tumefaciens (LBA4404) containing either
pMMACEipt or pGA643/ACE1.6/4MT-40GUS, using standard protocols
(Shillito and Saul, 1988). Plants were regenerated on solid
Murashige-Skoog medium containing 150 mg L1
kanamycin, but without CuSO4. Plants that
produced a positive result in the neomycin phosphotransferase test
(Herrera-Estrella and Simpson, 1988) were subcultured every 4 to 6 weeks and multiplied to produce clonal lines.
Gene Induction
Cu Treatment in Vitro
Tobacco transformants were treated with
CuSO4 (to a final concentration of 5, 10, or 50 µm) via the application of a liquid stock solution to the growth medium. This was allowed to soak in and
the plants were maintained, without subculture, while they were
monitored for physiological change.
Cu Treatment in Vivo
A completely randomized design was used for the in vivo experiment
with the treatment structure consisting of Cu2+
applied to two tobacco strains (Cu-GUS and Cu-ipt), each
having two to four independently transformed lines and each line having four to seven clonal plants. The experimental unit was a single plant.
Each plant was removed from the solid Murashige-Skoog medium and placed
in sterilized pumice. The plants were grown during a 16-h photoperiod
with a nominal photon flux density of 700 µmol m2 s1 and a day/night
temperature regime of 24/21°C. They were watered when required
with Murashige-Skoog medium and exposed to Cu2+
from the beginning of the experiment. Root tissue was removed throughout the experiment for GUS analysis. Thirty days after planting
out, the following variables were measured for each plant: plant
height, number of leaves, number of lateral buds (greater than 3 cm in
length), length of lateral bud (measured from the base to the tip of
the largest leaf), number of leaves in each lateral bud (not including
those protecting the meristem), and position of each lateral bud (node
number). The SAS Institute (Cary, NC) computer software package was
used to fit a general linear model to each variable, and the effects
due to strain and line (within each strain) were tested for
significance.
Fluorogenic GUS Assay
Fluorogenic GUS assays were performed as described by Jefferson
(1987). Protein content was determined according to the work of
Bradford (1976), and GUS activity was expressed in picomoles per minute
per milligram of protein.
Cytokinin Analysis
Harvested tissue was weighed and placed in modified Bieleski
solution (Jameson et al., 1987) at 
20°C until needed. The tissue was homogenized on ice, and internal standards
([3H]ZRTA, [3H]iPATA,
and [14C]AMP [Amersham]; 30,000 cpm each)
were added before storage at 4°C for 48 h. The extract was
centrifuged, the supernatant removed, and the pellet resuspended in
Bieleski solution for 24 h. The extract was centrifuged again and
the second supernatant was combined with the first.
The cytokinins were purified by passage through linked columns of
polyvinylpolypyrrolidone (Sigma; Palni et al., 1983), DEAE-cellulose (DE52, Whatman), and octadecyl silica (Bondesil, Analytichem
International, Boston, MA; Jameson and Morris, 1989), which had been
preconditioned with 10 mm ammonium acetate (pH 6.5; H. Zhang, personal communication). The columns were washed with ammonium
acetate and the polyvinylpolypyrrolidone column was discarded. The
nucleotides were eluted from the DEAE-cellulose column with 1 m acetic acid, and the free bases, ribosides, and glucosides were eluted from the octadecyl silica column with methanol.
Bulk separation of the free base/riboside fraction from the glucosides
was achieved using normal-phase HPLC on an Alphasil 5NH2 column (250 × 4.6 mm, HPLC Technology,
Cheshire, UK). The individual cytokinin forms were separated using
reverse-phase HPLC on an octadecyl silica column (5 µm, 250 × 4.6 mm; Altex, Berkley, CA). Both separations were described by Lewis
et al. (1996).
Before HPLC separation the nucleotide and glucoside fractions were
converted to their riboside and/or free base forms (Lewis et al.,
1996) and additional cytokinin standards
([3H]ZRTA and [3H]iPATA
[10,000 cpm]) were added.
Two antibody clones were used for radioimmunoassay: clone 16, which had
good affinity for hydroxylated cytokinins such as Z, DZ, ZR, DZR, and
Z9G, and clone 12, which cross-reacted with iP, iPA (Trione et al.,
1985), and iP-9-G (Lewis et al., 1996). Radioimmunoassays were carried
out as described by Jameson and Morris (1989). The antibodies were
diluted in radioimmunoassay buffer so that 50 µL bound 50% of the
[3H]trialcohol in the absence of competitive
antigen. Nonspecific binding was low for all assays. Aliquots from each
HPLC fraction were evaporated to dryness and 5000 cpm of
[3H]ZRTA or [3H]iPATA
was added with the radioimmunoassay buffer. Fractions 1 to 60 (which
contained the hydroxylated forms) were assayed with clone 16, and
fractions 51 to 80 (which contained the nonhydroxylated forms) were
assayed with clone 12. Standard curves of ZR (clone 16) and iPA (clone
12) were conducted in triplicate with every radioimmunoassay.
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RESULTS |
Morphology of Tobacco Transformants in Tissue Culture
Thirty-one independent transformants (Cu-ipt plants)
were produced following transformation of tobacco with the
ipt gene linked to the Cu-controllable promoter.
Transformations using similar vectors containing the GUS reporter gene
in place of the ipt gene produced 11 independent
transformants (Cu-GUS plants). The transformants were propagated in
tissue culture to produce clonal lines.
Twenty-eight of the Cu-ipt lines grew in vitro in a manner
identical to the Cu-GUS controls. The growth pattern of these plants, which consisted of a single, apically dominant shoot with no visible lateral buds, is shown in Figure 1, left.
These plants produced abundant root systems within 4 weeks of growth in
the presence of kanamycin.
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| Figure 1.
Morphological comparison of tobacco lines ID8,
ID9, and IR19. Left, Morphologically normal line ID8 21 d after
subculture. Top right, Morphologically aberrant line ID9 21 d
after subculture. Bottom right, Morphologically aberrant line IR19
21 d after subculture. The plants were grown in tissue culture on
solid Murashige-Skoog medium containing 150 mg L1
kanamycin but no CuSO4.
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Three of the Cu-ipt lines grew in a manner consistent with
aberrant cytokinin expression: ID9 initially produced an apically dominant shoot but failed to form roots and produced callus at the base
of its stem. Shoot elongation was limited and lateral buds appeared in
the axils of the leaves. Although these buds did not elongate,
additional buds continued to emerge. This pattern continued until ID9
became a mass of miniature leaves growing on a lump of callus (Fig. 1,
top right). This phenotype was stable in culture. IR19 was similar to
ID9 but grew wrinkled leaves that produced epiphyllic shoots and had a
tendency toward early necrosis (Fig. 1, bottom right). IR13 also showed
reduced apical dominance, grew callus from the base of its stem, and
produced small leaves (not shown).
Southern Analysis of the Tobacco Transformants
Southern analyses were carried out on five of the independent
transformants. The analysis included both morphologically normal and
morphologically aberrant lines. Genomic DNA was digested with EcoRI, which is known to have no restriction sites within
the T-DNA of pMMACEipt, an area approximately 5 kb in size. The results confirmed the presence of the ipt gene in all of the lines
analyzed (data not shown).
Comparison of Cytokinin between Normal and Aberrant
Transformants
The cytokinin content of the aberrant line ID9 was analyzed to
confirm that its altered morphology was correlated with cytokinin overproduction. A line that was morphologically normal in tissue culture and shown via Southern analysis to be transformed (ID8) was
included in the analysis for comparison.
Line ID9 showed a marked increase in cytokinin content over line ID8
(Table I). In ID9 tissue ZR and iPA,
ZOG and ZROG, and ZNT were detected. The most abundant cytokinin in
line ID9 was ZR at 134.4 pmol g1 fresh weight,
making up 42% of the total cytokinin detected. The combined glucosides
ZOG and ZROG (96.4 and 46.9 pmol g1 fresh
weight, respectively) contributed an equivalent amount, with smaller
quantities of ZNT and iPA being detected. By comparison, line ID8
produced only trace quantities of DZR, iPA, ZOG, and ZNT. None of these
was detected at a level large enough to allow accurate quantification
(Table I). In addition to the cytokinin forms shown in Table I, the
experimental system would have detected Z, DZ, iP, Z9G, iP9G, DZOG,
DZROG, DZNT, and iPNT if they had been above the detection limit of the
assay.
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Table I.
Cytokinins detected in the leaf tissue of Cu-ipt
tobacco lines ID8 and ID9 under noninductive conditions
Values have been corrected for losses during purification and for
differential cross-reactivity of the antibodies. The detection limit
for ZR with clone 16 was 0.6 pmol and for iPA with clone 12 it was 0.8 pmol.
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Physiological Changes in Morphologically Normal Plants in
Tissue Culture following ipt Gene Induction
Two in vitro experiments were performed on morphologically normal
transformants to assess the impact of cytokinin production on plant
physiology. In the first experiment we investigated the morphological
alteration of Cu-ipt and Cu-GUS plants following treatment
with varying concentrations of CuSO4. Following
97 d of treatment with 5, 10, or 50 µm
CuSO4, the leaves of the Cu-ipt plants
were clearly greener than those of the Cu-GUS plants. This trend was
most obvious in plants treated with 50 µm
CuSO4 (Fig. 2). The
second in vitro experiment compared the cytokinin profile of
Cu-ipt plants from the same line treated with either 50 µm CuSO4 or water. Following 3 months of treatment, most of the plants were not senescent. However,
leaf tissue was harvested for cytokinin analysis based on the
senescence time frame observed in the first experiment.
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| Figure 2.
Comparison of leaf senescence in Cu-GUS and
Cu-ipt tobacco transformants following treatment with 50 µm CuSO4. Cu-GUS (left) and
Cu-ipt (right) plants growing in vitro following 97 d of treatment with 50 µm CuSO4.
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Cytokinin analysis showed that one of the Cu-ipt lines
produced a marked increase in cytokinin content following
CuSO4 treatment compared with uninduced tissue of
the same line (Table II). In the
CuSO4-treated tissue the cytokinin free bases
showed the largest increase, with Z measuring 27.5 pmol
g1 fresh weight and iP measuring 40.5 pmol
g1 fresh weight. DZR showed the largest
increase of any single cytokinin form (52.2 pmol
g1 fresh weight), and a smaller quantity of ZOG
(11.8 pmol g1 fresh weight) was also observed.
By comparison, only a trace of iP was detected in uninduced tissue
(Table II). In addition to the cytokinin forms shown in Table II, the
experimental system would have detected DZ, ZR, iPA, Z9G, iP9G, DZOG,
ZROG, DZROG, ZNT, DZNT, and iPNT had these been above the detection
limit of the assay.
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Table II.
Comparison of the cytokinins detected in the leaf
tissue of Cu-ipt tobacco line ID13 following in vitro treatment with
water (CuSO4) or Cu (+CuSO4).
Values have been corrected for losses during purification and for
differential cross-reactivity of the antibodies. The detection limit
for ZR with clone 16 was 0.6 pmol and for iPA with clone 12 it was 0.8 pmol.
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Morphological Changes in Whole Plants following ipt
Gene Induction
To determine the effect of a controlled endogenous cytokinin
increase on whole plant morphology, we treated morphologically normal
Cu-ipt and Cu-GUS strains with CuSO4
in vivo. Six independently transformed lines were included: two Cu-GUS
control lines and four Cu-ipt lines. Each line was
represented by 4 to 7 plants, resulting in a total of 31 plants over
the whole experiment. After being removed from tissue culture, all
plants required an initial recovery period of 1 week, after which they
began rapid stem elongation and leaf expansion. After 17 d of
exposure to Cu2+, the Cu-ipt plants
began to display lateral bud growth. This continued until the
conclusion of the experiment (Fig. 3),
when the growth pattern of each plant was measured.
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| Figure 3.
Comparison of growth patterns in Cu-GUS and
Cu-ipt tobacco transformants following treatment with
Cu. Left, Representative plant from Cu-GUS line GD11 following 30 d of treatment with Cu. Middle and right, Representative plants from
Cu-ipt line ID8 following 30 d of treatment with
Cu.
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There were a number of significant differences in the growth pattern of
the Cu-ipt plants compared with the control plants, which
are given in Table III. The
Cu-ipt plants had a greater number of lateral buds (P < 0.0001), a greater lateral bud length (P < 0.001), a greater
number of leaves per lateral bud (P < 0.01), and a greater leaf
number per plant (P < 0.0001). Stems had also appeared on some of
the lateral buds of the Cu-ipt plants (6 of 59), whereas
they were completely absent from the controls. Measurements that were
not significantly different between the Cu-ipt and Cu-GUS plants included plant height and the node number from which the lateral
buds grew.
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Table III.
Growth patterns of Cu-GUS and Cu-ipt tobacco from
the in vivo experiment following 30 d of exposure to Cu
Data presented are the estimates of the least-squares means of the
morphological characteristics measured. ses are in
parentheses. Those figures in each column followed by a different
letter are significantly different at P  0.05.
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During the experiment root samples were taken from the Cu-GUS
lines for analysis of GUS expression to confirm that the
Cu-controllable promoter was directing expression in the roots. These
plants displayed strong GUS expression (1376.0-5298.2 pmol
min1 mg1 protein) in
the root tissue.
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DISCUSSION |
We regarded the Cu-controllable gene expression system as an
attractive candidate for use in the control of ipt gene
expression, mostly because of the tight temporal control it exhibits
(Mett et al., 1993). Because even small increases in endogenous
cytokinin concentration have been shown to have a significant effect on plant growth and development (Medford et al., 1989; Smart et al., 1991;
Smigocki, 1991), it was important that the lowest possible background
be maintained before gene induction. Another advantage of using this
system comes from the previous observation that, when its expression
was controlled by the 46-bp TATA sequence of the CaMV 35S promoter, GUS
activity was detected only in the roots of tobacco plants (Mett et al.,
1996). Therefore, the expression of the introduced ipt gene
could be expected to occur in the roots and to supplement the naturally
originating cytokinin.
A Grossly Aberrant Morphology Results from Uncontrolled Cytokinin
Expression
Despite the fact that most plant lines grew normally, three lines
(ID9, IR13, and IR19) showed marked morphological differences in tissue
culture when compared with controls. Morphological changes included the
formation of small, rounded leaves that did not expand, breaking of
apical dominance, lack of root formation, callus formation at the stem
base, and dwarfing (Fig. 1). These features are indicative of cytokinin
overproduction and have been repeatedly described by groups working
with ipt genes (Medford et al., 1989; Smigocki, 1991;
Beinsberger et al., 1992; Li et al., 1992; Hewelt et al., 1994).
Additional features displayed only by line IR19 have also been
previously described, including the production of wrinkled leaves that
showed premature necrosis and epiphyllic shoot production (Estruch et
al., 1991; Li et al., 1992; Hewelt et al., 1994).
The morphological changes we observed in these lines indicated that the
ipt gene sequence was present and functional. The cytokinin
profile of one of the aberrant lines (ID9) confirmed that the
concentrations of five cytokinin forms had increased over those
observed in a morphologically normal line (ID8; Table I). The presence
of the physiologically active ribosides in line ID9 was not unexpected
in light of its altered morphology. However, of particular interest was
the presence of the O-glucosides. Regarded as storage forms,
the O-glucosides are probably used to reduce the
concentration of active cytokinin within the plant (for review, see
Jameson, 1994). This seems especially likely in the case of ZROG, since
ZR was detected as the most prominent cytokinin in ID9 tissue. Redig et
al. (1996) also suggested that O-glucoside conjugation
occurs in ipt-expressing tissue. As in the work reported here, they detected little conversion to the N-glucoside
forms.
The increase in ZNT seen in line ID9 reinforces the importance of
analyzing all of the cytokinin forms. ZNT is one of the first forms
produced following the formation of iPNT by the enzyme isopentenyl
transferase and may act as a source for the formation of ZR by
phosphatase breakdown. The use of modified Bieleski solution (Jameson
et al., 1987) for tissue storage following harvest was expected to stop
the nonspecific cleavage of nucleotides by phosphatase enzymes.
However, the adjustment of sample pH using concentrated ammonia before
its application to chromatography columns is suspected to have caused
some breakdown of the nucleotides (H. Zhang, personal communication),
which would have added to the ZR pool detected in this tissue.
The cytokinin analysis of line ID9 confirmed that the ipt
gene was still able to produce a functional isopentenyl
transferase, although the expression of the ipt gene was
uncontrolled in lines ID9 and IR19. This could have been due to
position effect and/or copy number (Gendloff et al., 1990). The
presence of plant lines that were transformed with the ipt
gene but were morphologically indistinguishable from the Cu-GUS
controls indicated that in these lines expression of the ipt
gene was under tight control.
Controlled Cu-ipt Expression in Tissue
Culture Results in Delayed Leaf Senescence
The cytokinins are believed to be involved in the regulation of
senescence in leaves. The exogenous application of cytokinin to leaf
tissue has been shown to delay its senescence (Richmond and Lang, 1957;
Noodén et al., 1979), and cytokinin levels have been observed to
decline in senescing leaf tissue (Singh et al., 1992). A number of
groups working with ipt genes have described delayed leaf
senescence in their transformants (Smart et al., 1991; Hewelt et al.,
1994; Gan and Amasino, 1995).
To determine the influence of endogenously produced cytokinin over leaf
senescence in our transformants and the level of control exerted by the
Cu-controllable promoter over ipt gene expression, Cu-ipt and Cu-GUS lines were treated in vitro with either
CuSO4 or water. Following
CuSO4 treatment one line displayed a clear increase in cytokinin content (Table II). A total of 132.0 pmol g1 fresh weight was detected, compared with
only trace amounts of cytokinin following water treatment. This result
provided conclusive evidence that the expression of the ipt
gene was under the tight control of the Cu-controllable system to which
it had been fused.
The increase in the concentration of Z following
CuSO4 application is particularly interesting.
The work of Singh et al. (1992) showed that Z was the most abundant
cytokinin in nonsenescent tobacco leaves and that its concentration
declined when senescence began to occur. As suggested in reference to
the overexpressing line ID9, the detection of the
O-glucoside group ZOG in the Cu-treated tissue may be
indicative of an attempt to lower the levels of the very active Z base.
Despite the number of groups that have observed delayed leaf senescence
as a major physiological response to ipt gene expression, only Smart et al. (1991) undertook detailed cytokinin analysis of this
tissue. This is unfortunate since a detailed comparison of the
cytokinin levels and forms required to delay senescence would be of
interest, especially in systems that show tightly controlled
morphology, such as that reported by Gan and Amasino (1995).
In their analysis of tobacco transformed with an ipt gene
controlled by a heat-shock promoter, Smart et al. (1991) treated areas
of leaves attached to the plant with 42°C for 2 h. Analysis of
the treated regions and comparison with untreated regions showed marked
increases in Z (13.4-fold), ZR (8-fold), iP (7.8-fold), DZ (8.1-fold),
and DZR (7.5-fold). The nucleotide forms ZNT, iPNT, and DZNT also
increased, and a small increase was observed in iPA. Unfortunately, the
glucoside forms were not included in the analysis by Smart et al.
(1991). The concentration of Z (120.6 pmol g1
fresh weight) was markedly higher than that reported here, the concentration of iP was similar, and the riboside DZR was markedly lower (3.0 pmol g1 fresh weight). It is
possible that these differences could be due to differences in the
gene-induction method (heat shock versus Cu application), plant growth
conditions (in vivo versus in vitro), or the tissue of origin of the
cytokinins (potentially every cell versus root tissue only).
Nevertheless, all of the forms seen to increase in our work were also
observed to increase by Smart et al. (1991). The most significant
difference between the two sets of data is the amount of cytokinin
production by transformed but uninduced leaf material. Smart et al.
(1991) detected a significant increase in the concentration of
cytokinin in tissue that had not been heat shocked. It is possible that
some of the increase may have been due to cytokinin transport from
heat-shocked areas to non-heat-shocked areas. However, the observation
of morphological changes in untreated whole plants indicates that some
expression occurred in the absence of heat shock. The increase in total
cytokinin was 13-fold that seen in untransformed leaf tissue (averaged
over all groups detected). Even when more tightly controlled promoters have been used to control ipt gene expression, e.g.
tetracycline-inducible promoter (Redig et al., 1996; Faiss et al.,
1997), some cytokinin production has been reported in the noninduced
state. In contrast, cytokinins from the Cu-ipt line could
not be detected in uninduced leaf tissue (except for a trace level of
iP), and this pattern was identical to that of the Cu-GUS controls
(data not shown).
Morphological alteration due to ipt gene induction was
clearly observable in the first tissue culture experiment. Tobacco from
the Cu-ipt and Cu-GUS lines were treated with 5, 10, or 50 µm CuSO4 and
visually monitored for morphological changes. At the end of 3 months,
all of the Cu-ipt plants showed delayed leaf senescence
compared with the Cu-GUS controls. However, this response was most
obvious in plants treated with 50 µm
CuSO4 (Fig. 2), which is consistent with the
previous observation of Mett et al. (1993) that the quantity of
CuSO4 applied influences the level of gene
expression.
Controlled Cu-ipt Expression in Whole
Plants Results in a Break in Apical Dominance
Morphological alteration was also observed in the in vivo
experiment. Four independently transformed Cu-ipt lines were
included, with each line represented by at least four clonal
replicates. Measurements made during the experiment allowed accurate
comparison between the growth patterns of the lines, with particular
attention paid to lateral bud growth. The results show that the
Cu-ipt lines had significantly different morphology compared
with Cu-GUS controls (Table III) after 30 d of exposure to
Cu2+. Whereas the controls displayed strong
apical dominance, the Cu-ipt plants displayed a clear
release of lateral buds from apical dominance (Fig. 3). This was
confirmed by significant increases in lateral bud number, lateral bud
length, lateral bud leaf number, and total plant leaf number (Table
III), and the presence of stems on some of the lateral buds.
With the exception of Van Loven et al. (1993), to our knowledge there
are no other reports that document the extensive physiological data
required for the statistical analysis of growth patterns of
ipt-transformed plants. Using their data to analyze the
effect of endogenous cytokinin production on plant growth when
ipt expression was controlled by a heat-shock promoter, Van
Loven et al. (1993) showed that heat shock itself interfered with whole
plant growth, reducing both internode length and stem diameter.
Furthermore, they suggested that in vitro cultivation, a stressful
situation for plant growth, activated the heat-shock promoter, thereby
inducing transient cytokinin production before heat shock. This may
explain the pre-heat-shock expression of ipt genes in the
work of Medford et al. (1989), Smart et al. (1991), and Smigocki
(1991). In light of the above complications, recording physiological
data for statistical analysis appears to be necessary.
The release of lateral bud growth from apical dominance is the most
consistent morphological effect displayed by whole plants transformed
with ipt genes (Medford et al., 1989; Smart et al., 1991;
Smigocki, 1991; Van Loven et al., 1993; Hewelt et al., 1994; Faiss et
al., 1997). This aspect of development is also inducible by exogenous
cytokinin application (Sachs and Thimann, 1964). Under normal
conditions lateral bud growth in tobacco occurs only after flowering
and at the most apical buds. However, in ipt transformants lateral bud release has been observed to occur in vegetative plants, either around the middle nodes (Van Loven et al., 1993) or from the
base to mid-node (Hewelt et al., 1994), a phenomenon that is shown
in Figure 3.
Spatial ipt Gene Expression Results in Controlled
Physiological Changes
A number of other morphological changes have been
observed by various groups working with the ipt gene. These
include the reduction of root mass (Medford et al., 1989; Smigocki,
1991; Van Loven et al., 1993), reduction of plant height (Medford et al., 1989; Smart et al., 1991; Smigocki, 1991; Van Loven et al., 1993),
increased stem thickening (Ainley et al., 1993; Van Loven et al.,
1993), and chlorosis and wrinkling of the leaves (Ainley et al., 1993).
None of these changes was observed in the work reported here; the
ipt transformed plants appeared to be identical to controls
except in respect to apical dominance and leaf senescence. This is
particularly interesting considering the spatial control exerted by the
Cu-controllable gene expression system over gene expression (Mett et
al., 1996). Cytokinin production from the plants studied here was
targeted to the roots, which are believed to be a key site of cytokinin
biosynthesis in the whole plant (Letham, 1994). Conversely, in many of
the plants previously transformed with inducible ipt genes
(e.g. those controlled by heat-shock promoters), expression was not
targeted to a particular tissue but occurred throughout the plant,
resulting in the aberrant morphology described above. In fact, many of
the features described by these groups, such as wrinkling and chlorosis
of the leaves, reduction of plant height, and reduction of root mass,
were seen in our experiments only in lines that did not have controlled
ipt expression (ID9, IR13, and IR19).
Our data support the classic notion of root-derived
cytokinin influencing leaf senescence and apical dominance. However,
Faiss et al. (1997) utilized the bacterial tetracycline
repressor-operator complex in association with the ipt gene,
and concluded that released apical dominance and delayed senescence
were a consequence of localized cytokinin biosynthesis and were not due
to enhanced cytokinin production by induced root tissue. Although
increased ZR (> 10-fold) was noted in the transpiration stream, the
subsequent break in apical dominance and delayed leaf senescence of
whole plants were ascribed to tetracycline movement from the roots. To
distinguish between enhanced cytokinin export from the roots and the
movement of tetracycline, reciprocal grafts were carried out between
wild-type and transgenic tissue. Neither lateral shoot growth nor
senescence of wild-type material was affected by the presence of
induced transgenic rootstock. However, the presence of massive shoot
proliferation on the ipt rootstock itself may have created a
large metabolic sink. This could have arisen because of a lack of
cytokinin translocation through the graft union. Faiss et al. (1997)
noted that cytokinin levels in the xylem were normal beyond the graft
union.
In this work we produced transgenic tobacco carrying the ipt
gene under the control of the Cu-inducible gene expression system. The
temporal and spatial levels of control exerted by this system have
allowed the production of physiologically normal plants that can be
induced to produce more cytokinin at a desired time. This system will
be used to study the role of the endogenous cytokinins in plant growth
and development and to resolve the issue of cytokinins as long-distance
signals.
| |
FOOTNOTES |
1
Present address: Department of Plant Biology and
Biotechnology, Massey University, Private Bag 11222, Palmerston North,
New Zealand.
*
Corresponding author; e-mail mmckenzie{at}hort.cri.nz; fax
64-6-350-5694.
Received September 2, 1997;
accepted November 26, 1997.
2
Now incorporated into The Institute of Molecular
Biosciences, College of Science, Massey University, Palmerston North,
New Zealand.
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
DZ, dihydrozeatin.
DZNT, DZ nucleotide.
DZOG, DZ-O-glucoside.
DZR, DZ riboside.
DZROG, DZ
riboside-O-glucoside.
iP, isopentenyladenine.
iP9G, isopentenyl-9-glucoside.
iPA, isopentenyl adenosine.
iPATA, iPA
trialcohol.
iPNT, isopentenyl nucleotide (isopentenyl AMP).
ipt, isopentenyl transferase gene.
Z, zeatin.
Z9G, Z-9-glucoside.
ZNT, Z nucleotide (Z riboside-5
-monophosphate).
ZOG, Z-O-glucoside.
ZR, Z riboside.
ZROG, Z
riboside-O-glucoside.
ZRTA, Z riboside trialcohol.
| |
ACKNOWLEDGMENTS |
The authors wish to thank Leesa Lochhead for technical
assistance and Dr. Nihal De Silva for statistical expertise. The
cytokinin antibodies were a generous gift to P.E.J. from Prof. R.O.
Morris (University of Missouri, Columbia).
| |
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Abstract
Introduction