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Plant Physiol. (1998) 118: 773-781
Overexpression of 20-Oxidase Confers a Gibberellin-Overproduction
Phenotype in Arabidopsis
Shihshieh Huang*,
Anuradha S. Raman,
Joel E. Ream,
Hideji Fujiwara,
R. Eric Cerny, and
Sherri M. Brown
Plant Growth and Development Group, Monsanto Company, 700 Chesterfield Parkway North, St. Louis, Missouri 63198
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ABSTRACT |
In the gibberellin (GA) biosynthesis
pathway, 20-oxidase catalyzes the oxidation and elimination of
carbon-20 to give rise to C19-GAs. All bioactive GAs are
C19-GAs. We have overexpressed a cDNA encoding 20-oxidase
isolated from Arabidopsis seedlings in transgenic Arabidopsis plants.
These transgenic plants display a phenotype that may be attributed to
the overproduction of GA. The phenotype includes a longer hypocotyl,
lighter-green leaves, increased stem elongation, earlier flowering, and
decreased seed dormancy. However, the fertility of the transgenic
plants is not affected. Increased levels of endogenous GA1,
GA9, and GA20 were detected in seedlings of the
transgenic line examined. GA4, which is thought to be the
predominantly active GA in Arabidopsis, was not present at increased
levels in this line. These results suggest that the overexpression of
this 20-oxidase increases the levels of some endogenous GAs in
transgenic seedlings, which causes the GA-overproduction phenotype.
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INTRODUCTION |
During developmental transitions, differentiation, or response to
environmental changes, plants regulate their growth by varying the
levels of endogenous phytohormones. One class of phytohormones, GAs,
are found in young tissues of the shoot and developing seeds, and have
regulatory roles in seed germination, stem elongation, flowering, and
fruit set (for review, see Crozier, 1983 ). Whereas tissue
responsiveness is an important factor that also contributes to
GA-mediated developmental processes, these processes are directly responsive to the concentration of bioactive GAs present.
It is believed that de novo biosynthesis is the main source of
bioactive GAs in growing tissues. Thus, enzymes involved in GA
biosynthesis are likely to be the regulators of GA-related growth and
can serve as targets for the manipulation of plant growth and
development through genetic engineering. In Arabidopsis there are at
least five loci involved in GA biosynthesis: GA1, GA2, GA3, GA4, and
GA5. These loci were identified in mutants that have a GA
requirement for normal growth (Koornneef and van der Veen, 1980). By
quantifying endogenous GAs and applying various GAs and GA precursors
to these mutants, each GA locus has been assigned an enzymatic function
in the GA biosynthetic pathway (for review, see Finkelstein and
Zeevaart, 1984). The first committed reaction of the GA biosynthesis
pathway is the cyclization of geranylgeranyl pyrophosphate to
ent-kaurene, a two-step conversion. Copalyl diphosphate
synthase, formerly ent-kaurene synthetase A, the enzyme
responsible for the first part of the reaction, is encoded by the
GA1 locus and has been cloned (Sun et al., 1992; Sun and
Kamiya, 1994). The GA2 locus encodes ent-kaurene
synthase, which completes the conversion of geranylgeranyl
pyrophosphate to ent-kaurene (Yamaguchi et al., 1998).
Plants bearing mutations in the GA3 locus show a growth
response only to intermediates after ent-kaurenal in the GA
biosynthesis pathway. Therefore, the GA3 locus probably
encodes a Cyt P450 monooxygenase, which catalyzes the oxidation of
ent-kaurene to ent-kaurenoic acid. The endogenous
concentration of various GAs in ga4 and ga5
mutant plants was carefully measured (Talon et al., 1990), and the
results suggested that GA5 and GA4 encode GA
20-oxidase and 3 -hydroxylase, respectively. Both genes have been
cloned, and GA5 protein produced in vitro exhibits GA 20-oxidase
activity (Chiang et al., 1995; Xu et al., 1995).
Oxidation at carbon-20 of GAs is thought to be an important
aspect of regulation in the GA biosynthetic pathway. In spinach the enhanced oxidation activity is associated with the bolting response
brought on by exposure to long days (Gilmour et al., 1986). In maize
seedlings evidence suggests that the 20-oxidase activities are
down-regulated as a result of feedback control by GA (Hedden and
Croker, 1992). The cloning of the GA5 locus in Arabidopsis
allowed the study of the regulation of 20-oxidase at the molecular
level (Phillips et al., 1995; Xu et al., 1995), and these studies
showed that expression of GA5 increases when plants are
transferred from short-day to long-day conditions and decreases when
plants are treated with bioactive GA. Furthermore, there are at least
three different 20-oxidase genes in Arabidopsis, and their expression
patterns are differentially regulated (Phillips et al., 1995).
Therefore, the developmental and environmental regulation of 20-oxidase
gene expression appears to affect the level of endogenous GAs, which
influences plant growth.
To study the effects of 20-oxidase expression in vivo, we isolated a
full-length cDNA fragment from Arabidopsis seedling RNA by
reverse-transcriptase PCR and overexpressed it under the control of a
constitutive promoter in Arabidopsis. The transgenic plants that
overexpressed the 20-oxidase cDNA exhibited a "GA-overproduction" phenotype. This phenotype includes longer hypocotyl length,
lighter-colored leaves, longer and accelerated stem elongation, earlier
flowering, and decreased seed dormancy. We also examined the endogenous
GA levels of the transgenic plants. The results suggest that the phenotype is the result of increased endogenous GA levels caused by
overexpression of the 20-oxidase cDNA.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
Seeds of Arabidopsis ecotype Columbia were obtained from Lehle
Seeds (Tucson, AZ). Plants were grown in potting soil (Scotts, Marysville, OH) in a growth chamber at 24°C with a 16-h photoperiod (120 µE m 2 s1). In
the seedling growth and germination studies, seeds were surface
sterilized and germinated on Murashige and Skoog medium (M0404, Sigma)
supplemented with Suc (1%) and Mes (0.5 g L1)
at pH 5.8.
Cloning of 20-Oxidase cDNA and Transformation Plasmid
Construction
The PCR primers 5 -ATGGCCGTAAGTTTCGTAAC-3 and
5-TTAGATGGGTTTGGTGAGCC-3, located at the start and end of the
20-oxidase reading frame, were designed based on the reported sequence
of GA5 (Xu et al., 1995). These primers were used in a
reverse-transcriptase-PCR reaction to amplify a 1.1-kb cDNA fragment
from RNA extracted from 5-d-old Arabidopsis seedlings. The PCR product
was inserted into the TA cloning vector (Invitrogen, San Diego, CA) for
further manipulation. To detect any mutations generated by PCR, several independent reverse-transcriptase-PCR-amplified cDNAs were sequenced and compared. The consensus GA5 cDNA was then inserted into
a binary transformation vector for Agrobacterium
tumefaciens-mediated transformation into Arabidopsis. The
resulting plasmid, pMON29925, contains the 20-oxidase cDNA under
transcriptional control of an enhanced 35S promoter. The  90 to 342
region of the original 35S promoter (Fang et al., 1989) was duplicated
to produce the enhanced 35S promoter. Arabidopsis plants were
transformed according to the method of Bechtold et al. (1993).
Identification of T2 Plants Containing the
Transgene by PCR
Genomic DNA was isolated from a single rosette leaf disc using the
hexadecyltrimethyl-ammonium bromide protocol (Stewart and Via, 1993).
The same PCR primers used to prepare the cDNA were used to analyze the
genomic DNA of individual T2 plants for the presence or absence of the transgene. T2 plants
positive for the presence of the transgene were determined to be
hemizygous or homozygous for the transgene locus by the segregation
ratios of kanamycin-resistant T3 progeny;
T2 plants lacking the transgene and also showing
uniform kanamycin sensitivity in T3 progeny were selected as the isogenic wild-type controls.
Isolation of Genomic DNA and DNA Gel-Blot Analysis
Genomic DNA was isolated from 0.25 g of
T2 seedlings from each transgenic line tested as
described previously (Coleman and Kao, 1992). Genomic DNA (5 µg) was
digested with SpeI, separated on a 0.8% (w/v) agarose gel,
and transferred to a nylon membrane (Qiagen, Chatsworth, CA).
Prehybridization, hybridization, washing, and detection of the membrane
were conducted by using the nonradioactive DIG system from Boehringer
Mannheim following the manufacturer's protocols.
Isolation of Total RNA and RNA-Blot Analysis
Total RNA was isolated from 5-d-old T2
seedlings of each transgenic line tested. Only kanamycin-resistant
seedlings were used for RNA isolation by TRIzol Reagent (Life
Technologies) following the manufacturer's protocols. Total RNA
samples (20 µg) were electrophoresed on 1.2% (w/v)
agarose/formaldehyde gels and transferred to a nylon membrane
(Qiagen). The blot was analyzed by the same DIG system that
was used in the DNA gel-blot analysis.
Measurement of Endogenous GA Levels
Approximately 1 g fresh weight of Arabidopsis tissue (7-d-old
seedling) was frozen with liquid nitrogen and ground into a fine powder
with a mortar and pestle. The frozen powder was transferred to a glass
40-mL centrifuge tube and homogenized (Pro300D, Pro Scientific, Monroe,
CT) in 80% (v/v) methanol. Deuterated GA standards (17,17-d2-GA1,
17,17-d2-GA4,
17,17-d2-GA9, and
17,17-d2-GA20; L. Mander, Australian National
University) were added to a level of 0.2 ng/mL before homogenization.
The homogenate was filtered (Whatman no. 42 filter paper) and the
filtrate was added to a 6-mL C18 chromatography
column (Bakerbond spe, J.T. Baker). GAs were eluted with 4 mL of
80% (v/v) methanol and the methanol was evaporated under vacuum. The
remaining aqueous phase was adjusted to pH 3.0 with HCl and partitioned
three times against hydrated ethyl acetate. The combined ethyl acetate
fractions were evaporated under a vacuum, resuspended in 35% methanol
containing 0.05% (v/v) acetic acid, and filtered (0.25 µm, 25 mm
Nylon Acrodisc, Gelman Sciences, Ann Arbor, MI). The filtered extract
was injected onto a C18 reverse-phase column
(Xpertek Spherisorb ODS-2, 5 µm, 4.6 mm × 250 mm) and eluted at
a flow rate of 1 mL/min with a 40-min linear gradient from 35% to
100% (v/v) acidified methanol controlled by a gradient controller
(model 680, Waters).
One-milliliter fractions were collected and pooled according to the
expected retention of GAs of interest, as determined by previous
chromatography of tritiated standards for GA1,
GA4, and GA9 (obtained from
R. Pharis, University of Calgary, Alberta, Canada). Pooled HPLC
fractions were evaporated under a vacuum and resuspended in 300 µL of
methanol. Each sample (100 µL) was methylated with diazomethane
(10-20 µL) in a 1-mL glass vial at room temperature. Excess
diazomethane and its solvent were removed with a stream of nitrogen.
Each methylated sample (except GA9) received 1 µL of pyridine and 50 µL of
N,O-bis(trimethylsilyl)trifluroracetamide and
was heated at 70°C for 45 min. Excess
N,O-bis(trimethylsilyl)trifluroracetamide was
removed with a stream of nitrogen. An aliquot of each sample was
injected into a gas chromatograph for GC-selected ion monitoring-MS. The gas chromatograph was typically programmed from 100°C to 300°C at 10°C/min. The MS signal-peak-height method for endogenous GAs was
used, and the di-deuterated GAs were chosen for quantitation.
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RESULTS |
Transformation of Arabidopsis Plants with a 20-Oxidase cDNA Driven
by an Enhanced 35S Promoter
Primers were designed based on the reported Arabidopsis 20-oxidase
sequence from Xu et al. (1995). These primers were used to isolate a
20-oxidase full-length cDNA fragment from 4-d-old Arabidopsis seedling
RNA by reverse-transcriptase PCR. The sequence of the PCR-amplified
20-oxidase cDNA was identical to that of GA5 (accession no.
U20872) except for four base-pair changes. Three were silent
substitutions and one resulted in the conversion of Lys-310 to Glu.
These base-pair changes were unlikely to be mutations resulting from
PCR because they appeared in several independent PCR-amplified
20-oxidase cDNA sequences. One possible explanation for the
discrepancies is the difference in ecotypes of Arabidopsis used for the
DNA preparation. The GA5 sequence was isolated from the
Landsberg erecta ecotype.
The binary Ti plasmid (pMON29925) containing the 20-oxidase cDNA driven
by an enhanced 35S promoter was introduced into Arabidopsis via
A. tumefaciens-mediated vacuum infiltration (Bechtold et
al., 1993). Transgenic T1 lines were selected by
growth on kanamycin-containing medium. Among the 36 transgenic
T1 plants identified, 33 were considerably taller
than the control plants, which had been transformed with another binary
Ti plasmid containing GUS driven by the same promoter. All
of the 33 T1 plants showed phenotypic segregation in their T2 progeny. We randomly chose four
segregating T1 lines (25-1, 25-2, 25-3, and
25-24) for further studies.
Phenotypic and Molecular Characterization of Four Transgenic Lines
Approximately 30 T2 seeds from each of the
four T1 lines were planted, and leaf samples
collected from each T2 plant were tested for the
presence of the transgene by PCR. Each plant in the segregating
T2 population was placed into one of two groups, depending
on the presence or absence of the transgene. The physical characteristics were then measured and compared between these two
groups from each line. Table I summarizes
the data obtained from all of the T2 populations.
In all four T2 populations examined, the
T2 plants containing the transgene were taller
than their isogenic counterparts by at least 15% and up to 30%, as
seen in lines 25-2 and 25-3. The increased height of all four
transgenic lines is attributable to increased internode length. The
plants containing the transgene also had lighter-green leaves, which was more pronounced in lines 25-2 and 25-3.
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Table I.
Phenotypic evaluation of 20-oxidase-overexpressing
lines
T2 seeds from each of the four T1 lines were
planted, and leaf samples collected from each T2 plant were
tested for the presence of the transgene by PCR. Each plant from the
segregating T2 population was placed into one of two
groups, depending on the presence or absence of the transgene. The
physical characteristics were then measured and compared between these
two groups for each line. Data are means ± SE.
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As shown in Table I, the progeny segregation of all four transgenic
lines fit a 3:1 ratio, which suggests that they contain an active
insertion locus in the genome. This is confirmed by DNA gel-blot
analysis (Fig. 1A). When genomic DNA was digested with
a restriction enzyme, which cuts outside the T-DNA region, the blot
showed only two bands on each lane. The lower bands present in all
lines represent endogenous 20-oxidase genes (indicated by the arrow)
and the higher-Mr bands are the transgenes.
Three more bands appeared on each lane of the blot when a
less-stringent hybridization condition was used (data not shown); these
were most likely the other 20-oxidase genes described previously in Arabidopsis (Phillips et al., 1995). Although all four lines showed a
single insertion locus, lines 25-1 and 25-24 had much stronger signals
than the other two lines. This is most likely attributable to tandem
repeats of transgenes at the insertion locus in each of two lines; this
was verified by repeating the DNA gel-blot analysis with other
restriction digestions (data not shown).
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| Figure 1.
DNA and RNA gel-blot analysis of
20-oxidase-overexpressing plants. A, DNA gel blot containing
SpeI digests of genomic DNA (5 µg per lane) isolated
from transgenic lines 25-1, 25-2, 25-3, 25-24, and a nontransgenic
plant was hybridized with a 20-oxidase cDNA probe. The arrow marks a
lower common band that is probably the endogenous 20-oxidase gene. B,
RNA gel blot containing total seedling RNA (20 µg per lane) isolated
from transgenic lines 25-1, 25-2, 25-3, 25-24, and a nontransgenic
plant was hybridized with a 20-oxidase cDNA probe. On the bottom is an
ethidium bromide stain of an agarose gel that was loaded with the RNA
(1 µg per lane) used in the gel blot.
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Total RNA isolated from kanamycin-resistant T2
seedlings of four T1 lines was used for RNA
gel-blot analysis. The results show a strong correlation between the
amount of transgene mRNA accumulated and the GA-overproduction
phenotype (Fig. 1B; lines 25-2 and 25-3). When the blot was exposed for
longer periods of time, the more weakly expressing line, 25-1, began to
show a similar banding pattern to the other lines; however, endogenous
expression of 20-oxidase was still not detectable under these
conditions. The lines containing many copies of the transgene per locus
(25-1 and 25-24) had lower steady-state RNA levels than lines 25-2 and 25-3, which had fewer copies of the transgene. This may be attributable to gene silencing, a phenomenon that has been observed in other transgenic plant systems (Meyer, 1996).
In each line, seeds produced by each homozygous transgenic plant were
collected and pooled to obtain a homozygous transgenic T3 population. All of the studies described below
were based on the homozygous transgenic T3
population of each line, except for line 25-3, in which hemizygous
plants were used. The integration of the transgene in line 25-3 caused
a recessive albino mutation.
Elongated Hypocotyl in Transgenic Seedlings
Many plant species have abnormally elongated stems when treated
with exogenous GA. A comparable phenomenon was observed in transgenic
lines even at the seedling stage. Transgenic and wild-type seeds were
germinated on vertical plates, as shown in Figure
2A, so that their hypocotyl and root
lengths could be more easily measured. The seedlings were grown under
either 16 h of light or total darkness with or without GA in the
medium. All transgenic seedlings were taller than wild-type seedlings
when grown in the light. The hypocotyls of 7-d-old seedlings of lines
25-2 and 25-3 were approximately twice the length of the wild-type
hypocotyls (4.2 and 3.8 versus 2.0 mm), whereas the root lengths
remained the same (Table II). Both
transgenic and wild-type seedlings were responsive to exogenous
GA3, but the differences in hypocotyl length were
not as dramatic under these conditions. Root lengths of transgenic and
wild-type seedlings were not influenced by exogenous GA3. When seeds were germinated in the dark, the
hypocotyls did not show a significant difference between transgenic and
wild-type seedlings regardless of the presence or absence of
GA3 in the medium. The T3
generations of lines 25-2 and 25-3 retained the strong
GA-overproduction phenotype, whereas lines 25-1 and 25-24 displayed
only a slight phenotype.
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| Figure 2.
Promoted growth of 20-oxidase-overexpressing
plants. The pictures compare the growth of transgenic (right) and
wild-type (left) plants at 1 week (A), 3 weeks (B), 3.5 weeks (C), and
4 weeks of age (D). The plants shown are from the T3
generation of transgenic line 25-2. The transgenic plants are
homozygous for the transgene and the wild-type plants are their
nontransgenic siblings.
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Table II.
The effect of light and GA3 on 7-d-old
T3 seedlings
Transgenic and wild-type seeds were germinated on vertical plates, as
shown in Figure 2A. These seedlings were grown under either 16 h
of light or total darkness with (+) or without () GA3 (10 µM) in the medium. Data are means ± SE
(n < 30).
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Growth Cycle of Transgenic Plants
GAs have been shown to induce flowering, especially in rosette
plants (for review, see Zeevaart, 1983), and we tested whether plants
overexpressing 20-oxidase would flower earlier than their wild-type
counterparts. In experiments done in the T3
generation, transgenic plants of lines 25-2 and 25-3 bolted 4 to 5 d (18.5 and 18.3 versus 23.0 d) earlier and had fewer rosette
leaves than wild-type plants (Table III).
However, consistent with the vegetative data, plants of lines 25-1 and
25-24 were indistinguishable from wild-type plants. The addition of
exogenous GA3 accelerated flowering in transgenic
lines 25-1, 25-24, and wild-type plants, with these plants approaching
the floral-timing pattern of the 25-2 and 25-3 plants. Thus, the
addition of exogenous GA3 to wild-type plants can
phenocopy the phenotype observed in transgenic lines 25-2 and 25-3 for
both floral timing and hypocotyl length (Tables II and III).
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Table III.
The flowering time of transgenic plants under long
days
Transgenic and wild-type plants were grown in alternating periods of
16 h of light and 8 h of dark, and the number of days for
primary bolt to reach a height of 1 cm for each plant was recorded.
Data are means ± SE (n = 16).
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Observations of the overall pattern of growth in the
T3 generation of these 20-oxidase-overexpressing
plants revealed not only increased stature but also a shortened growth
cycle in comparison with wild-type plants. As indicated in Table
IV and Figure 2, height differences
between transgenic lines 25-2 and 25-3 and wild-type plants are obvious
throughout the entire growth cycle. Because the 25-2 and 25-3 lines
flower earlier than the wild-type plants, the differences in height
measurements by days were inflated. However, height measurements at the
terminal stage clearly indicated that lines 25-2 and 25-3 were
significantly taller than wild-type plants. Plants of lines 25-2 and
25-3 reach 47.2 and 49.5 cm at global proliferative arrest of the
inflorescence meristems (Hensel et al., 1994) in comparison with 40.2 cm for the wild-type plants. It is interesting that the time required
to reach these heights was 37.4 and 37.2 d for the plants of lines 25-2 and 25-3, respectively, versus 39.4 d for the wild type. In addition,
these transgenic plants had fewer tertiary inflorescence branches than
wild-type plants. These observations suggest that GA promotes primary
growth and accelerates development throughout the plant's life cycle, probably at the expense of reducing tertiary branches. Transgenic lines
25-1 and 25-24 grew similarly and responded to exogenous GA3 like wild-type plants. In summary, these
results suggest that the phenotype displayed by two lines of the
transgenic plants (lines 25-2 and 25-3) is inheritable, and similar to
that of wild-type plants, with elevated GA levels attributable to
exogenous application of GA3. The two other
transgenic lines (lines 25-1 and 25-24) lost their phenotype at the
T3 generation. However, this so-called "gene-silencing"
phenomenon is often observed in other transgenic plant systems (Meyer,
1996), especially in transgenic plants containing multiple copies of
transgenes and being carried through multiple generations.
Plants of lines 25-2 and 25-3 produced more seeds than plants of the
wild type and lines 25-1 and 25-24, which had lost their phenotype
(Table IV). The data seem to suggest that overexpressing 20-oxidase in
Arabidopsis plants increases the number of seeds produced. However,
when GA3 was applied to these plants, the effect on seed yields was inconclusive (Table IV).
Reduced Seed Dormancy in Transgenic Seeds
Although seed germination in Arabidopsis can be influenced by a
variety of extrinsic factors, GA biosynthesis is a requirement. Several
Arabidopsis mutants impaired in the GA biosynthetic pathway are unable
to germinate independently and need to be supplied with exogenous GA
(Koorneef and van der Veen, 1980). Freshly collected T4 seeds of line 25-2 and wild-type seeds were
air dried and stored at room temperature. A small portion of these
seeds was sown on germination medium at weekly intervals, and the
fraction that had germinated was recorded every 12 h (Fig.
3). After 1 week at room temperature,
transgenic seeds began to germinate 3 d after planting and 54%
germinated within 10 d. In contrast, wild-type seeds did not
germinate until 4 d after planting and only 22% germinated within
10 d. As the seeds aged, dormancy decreased, as expected.
Transgenic seeds consistently germinated better than wild-type seeds
until the seed had been stored for 7 weeks. At this time, wild-type
seeds germinated as well as transgenic seeds and both seeds had nearly
100% germination efficiencies a few days later. However, when plated
seeds were cold treated for 4 d before being incubated for
germination, both transgenic and wild-type seeds germinated well, even
with less than 1 week of storage at room temperature.
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| Figure 3.
Germination efficiency comparison of freshly
collected transgenic line 25-2 T4 and wild-type seeds.
Germination efficiencies of seeds collected from the T3
generation of transgenic plants (+) and wild- type () plants are
shown. Seeds were plated on medium for germination at 1 ( ), 2 ( ),
3 ( ), 4 ( ), and 7 (×) weeks after harvesting. Transgenic plants
are indicated by solid lines and wild-type plants are indicated by
dashed lines.
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Analysis of Endogenous GA Levels in Transgenic Seedlings
In Arabidopsis three parallel steps of 20-oxidation in the GA
biosynthesis pathway have been suggested to produce
GA4, GA9, and
GA20 (Talon et al., 1990; Zeevaart and Talon,
1992). Subsequently, GA20 can be
3-hydroxylated or GA4 can be 13-hydroxylated
to produce GA1 (Fig.
4). To help differentiate between these
biosynthetic routes, we measured endogenous concentrations of
GA1, GA4,
GA9, and GA20 in
T4 seedlings of line 25-2 and wild-type seedlings (Fig. 5). In 7-d-old plants there were
higher concentrations of GA1 and
GA20 in transgenic than in wild-type seedlings,
whereas the concentration of GA4 remained about
the same. These results have confirmed that 20-oxidase-overexpressing
seedlings have higher 20-oxidase activity, mainly in the early
13-hydroxylation pathway of the GA biosynthetic pathway (Fig. 4).
However, we cannot conclude that the 20-oxidase activity also increases
in the other pathways. The endogenous level of
GA9 increased slightly, but the level of
GA4 in the seedlings under these conditions did
not change (Fig. 5).
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| Figure 4.
Proposed three endogenous GA 20-oxidations in
Arabidopsis. Different derivatives of C20-GAs can be
oxidized by endogenous 20-oxidases to produce C19-GAs. The
early 13-hydroxylation pathway leads to GA20; the
non-3,13-hydroxylation pathway leads to GA9; and the early
3-hydroxylation pathway leads to GA4. GA9 can
be 13-hydroxylated and 3-hydroxylated to give GA20 and
GA4. GA4 can be 13-hydroxylated and
GA20 can be 3-hydroxylated to give to GA1.
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| Figure 5.
Endogenous GA levels in line 25-2 T4
and wild-type seedlings. Endogenous GA1, GA4,
GA9, and GA20 levels in 7-d-old seedling were
measured by GC-MS after GA extraction. The amounts are indicated by
nanograms of GA in 1 g of seedlings (fresh weight).
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DISCUSSION |
20-Oxidase activity and expression have previously been shown to
be regulated by environmental or physiological changes (Gilmour et al.,
1986; Hedden and Croker, 1992; Xu et al., 1995), suggesting that
oxidation at carbon-20 of GA is a key regulatory step in the GA
biosynthetic pathway. We have confirmed these results by overexpressing
the GA5 cDNA in Arabidopsis, which gives rise to a
GA-overproduction phenotype in transgenic plants. The phenotype includes elongated stems, early flowering, reduced seed dormancy, and
an accelerated growth cycle. This phenotype is similar to spy mutants, whose mutations turn on the GA signal pathway
constitutively (Jacobsen and Olszewski, 1993), and the wild-type
plants repeatedly treated with exogenous GA. These similarities
strongly suggest that the phenotype in 20-oxidase-overexpressing lines
is a result of elevated endogenous GA levels.
The transgenic GA-overproduction phenotype can be further enhanced by
the addition of exogenous GA, which is also observed in spy
mutants (Jacobsen and Olszewski, 1993). As shown in Table II,
transgenic plants exposed to exogenous GA3
exhibited further increased seedling hypocotyl length. However, unlike
spy mutants, which have greatly reduced seed set, the
20-oxidase-overexpressing transgenic plants are fully fertile and even
display an increase in seed set. This could be attributable to
differences between the response of floral tissues to constitutive
activation of the GA signal transduction pathway and their response to
elevated endogenous GA levels. Alternatively, the observed differences may be the result of reduced expression of the enhanced 35S promoter driving 20-oxidase in reproductive tissues.
Although the 20-oxidase-overexpressing plants accumulate more GA than
wild-type plants and display a GA-overproduction phenotype under normal
growth conditions, there was no obvious phenotypic difference between
them when they were grown under conditions known to increase GA
sensitivity in the plants. For example, when seedlings were grown in
the dark, there was little difference in hypocotyl length between
transgenic and wild-type seedlings either in the presence or absence of
exogenous GA in the medium (Table II). This may be attributable to the
saturation of the GA response in dark-grown seedlings, a hypothesis
supported by the observation that Arabidopsis phyB mutant
seedlings are more sensitive to GA (Reed et al., 1996). Presumably, the
endogenous GA of wild-type plants exerts a maximal effect on hypocotyl
elongation when the seedlings are grown in the dark, and the excess GA
produced in transgenic plants or supplied exogenously does not
exaggerate the extended hypocotyl phenotype. Similarly, freshly
collected transgenic seeds germinated better than wild-type seeds of
the same age, but no difference was apparent between transgenic and wild-type seeds stored at room temperature for more than 7 weeks or
cold treated for 4 d. It has been suggested that GA sensitivity is
independent of GA biosynthesis during Arabidopsis seed germination and
that aging and chilling increase responsiveness (Koornneef and Karssen,
1984). The 20-oxidase-overexpressing transgenic seeds accumulate more
GA and, therefore, are able to germinate at a less GA-sensitive stage,
when higher GA levels are required to stimulate the response. Once
seeds are ripened or stratified, their increased GA sensitivity allows
wild-type and transgenic seeds to germinate equally well (even though
there is more GA produced in transgenic seeds).
GA1 is detected in Arabidopsis shoots, but
GA4 is thought to be the primary active GA in
Arabidopsis (Talon et al., 1990; Zeevaart and Talon, 1992). Also,
Phillips et al. (1995) showed that when expressed in Escherichia
coli, 20-oxidases preferred GA12
(non-13-hydroxylated GAs) to GA53
(13-hydroxylated GAs). In our studies, we detected increased levels of
GA1 but not GA4 in the
plants of the 25-2 line. The accumulation of GA20
suggests that the overproduced GA1 in transgenic
plants is derived from 3-hydroxylation of GA20
rather than from 13-hydroxylation of GA4 (Fig.
4). This finding seems to indicate that the overexpression of this
20-oxidase cDNA isolated from seedlings promotes the 13-hydroxylation GA biosynthesis pathway in transgenic seedlings, and that the accumulation of GA1, the end product of that
pathway, causes the phenotype. However, with only one transgenic line
and four GAs analyzed, these results are preliminary. It is difficult
to characterize the kinetics of overall GA biosynthesis without
additional transgenic lines and endogenous GAs being analyzed.
The phenotype exhibited in these transgenic plants is of agronomic
interest, particularly the accelerated growth cycle that promotes
primary stem growth with no reduction in seed set. Such characteristics
may be useful, for example, in forest cultivation, in which the growth
rate and primary stem mass are of greater concern, or in areas in which
a rapid-cycling cultivar would allow multiple plantings and harvests.
In fact, application of GA has long been used as a way to increase the
economic value of agriculture (Carlson and Crovetti, 1990). Here we
provide an alternative approach to GA application by genetic
engineering.
In summary, our findings suggest that 20-oxidase is the rate-limiting
step in the GA biosynthesis pathway in Arabidopsis. We demonstrate the
feasibility of manipulating the endogenous GA levels through genetic
engineering. The phenotypes displayed by these transgenic plants not
only have interesting agricultural implications but are also useful for
basic GA research.
| |
FOOTNOTES |
*
Corresponding author; e-mail
shihshieh.huang{at}monsanto.com; fax 1-314-737-7670.
Received May 26, 1998;
accepted August 10, 1998.
| |
ACKNOWLEDGMENTS |
We thank Debbie Stone and Wendi Zumwalt for plant transformation
and Greg Heck for valuable comments on the manuscript.
| |
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Top
Abstract
Introduction