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Plant Physiol. (1999) 120: 73-82
Heterologous Expression of Arabidopsis Phytochrome B in
Transgenic Potato Influences Photosynthetic Performance and Tuber
Development1
Alexandra Thiele,
Michael Herold,
Ingo Lenk,
Peter H. Quail, and
Christiane Gatz*
Albrecht von Haller Institut für Pflanzenwissenschaften,
Untere Karspüle 2, Georg August Universität
Göttingen, 37073 Göttingen, Germany (A.T., M.H., I.L.,
C.G.); and and University of California (Berkeley)/United
States Department of Agriculture Plant Gene Expression Center, 800 Buchanan Street, Albany, California 98710 (P.H.Q.)
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ABSTRACT |
Transgenic potato (Solanum
tuberosum) plants expressing Arabidopsis phytochrome B were
characterized morphologically and physiologically under white light in
a greenhouse to explore their potential for improved photosynthesis and
higher tuber yields. As expected, overexpression of functional
phytochrome B caused pleiotropic effects such as semidwarfism,
decreased apical dominance, a higher number of smaller but thicker
leaves, and increased pigmentation. Because of increased numbers of
chloroplasts in elongated palisade cells, photosynthesis per leaf area
and in each individual plant increased. In addition, photosynthesis was
less sensitive to photoinactivation under prolonged light stress. The
beginning of senescence was not delayed, but deceleration of
chlorophyll degradation extended the lifetime of photosynthetically
active plants. Both the higher photosynthetic performance and the
longer lifespan of the transgenic plants allowed greater biomass
production, resulting in extended underground organs with increased
tuber yields.
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INTRODUCTION |
Plant growth, development, and metabolic activities are regulated
by a range of environmental factors, including light, which is of
central importance. Light is perceived by a variety of photoreceptors that control developmental processes such as germination,
photomorphogenesis, flowering, and senescence, as well as metabolic
processes such as photosynthesis and assimilate allocation. It has been
pointed out before that the agricultural productivity of crop plants
might be enhanced by overexpressing one of these central regulators (Smith, 1992 ), and phytochromes are promising candidates for such an
improvement (Robson et al., 1996).
Phytochromes represent a family of red-light-absorbing photoreceptors
that can exist in the physiologically inactive Pr form and the active
Pfr form (for reviews, see Quail et al., 1995; Smith, 1995; Casal et
al., 1998). Pr and Pfr are interconvertible by red or FR light,
respectively. This absorption profile is extremely useful for the
detection of shade or the presence of neighboring plants. At high
relative proportions of FR radiation, which occur under shade
conditions or in dense plant populations, the photoequilibrium is
shifted toward the inactive Pr form. Under these conditions, green
plants exhibit various symptoms of the shade-avoidance response, such as promotion of stem and petiole elongation, reduced leaf thickness, reduced chlorophyll synthesis, and increased apical dominance (Smith and Whitelam, 1997). The shade-avoidance
response reduces the availability of resources for storage and
reproduction.
Five PHY genes have been identified in
Arabidopsis that have 50% to 80% identity at the amino acid level
(Sharrock and Quail, 1989; Clack et al., 1994). The best-characterized
members are PHYA and PHYB. phyA accumulates in
the dark and is rapidly degraded upon conversion to the labile Pfr form
(Pratt et al., 1997). It is responsible for detecting continuous
FR light and dampens the shade-avoidance response under high relative
proportions of FR light (McCormac et al., 1992; Heyer et al., 1995). In
contrast, light-stable phyB is expressed at low but relatively constant levels in light- and dark-grown plants. Being sensitive for the detection of red light, phyB suppresses the shade-avoidance response under high relative proportions of red light.
Tobacco plants strongly overexpressing oat phyA (Keller et al.,
1989) show a light-exaggerated phenotype even under white light:
internodes are shorter, leaves are darker green, smaller in size, and
slightly thicker, and the axillary buds are under less apical control.
Because many genes involved in photosynthesis are coordinately
regulated by phytochrome, phyA overexpressors consistently express
higher amounts of several enzymes of carbon metabolism (Sharkey et al.,
1991). The latter result seemed to be promising with respect to
increasing the photosynthetic performance. However, unexpected physical
modifications of the chloroplasts worsened the productivity of these
plants (Sharkey et al., 1991). Lower-expressing lines do not show the
light-exaggerated phenotype under white light. Robson et al. (1996)
nevertheless demonstrated that the harvest index of these low phyA
overexpressors was positively affected when they were planted at high
densities. Under these conditions the shade-avoidance response was
suppressed because of the higher sensitivity of these plants to FR
light reflected by neighboring plants. Thus, assimilates were allocated
to leaves rather than to stems. These data indicate that the
performance of crop plants might be improved by altering phytochrome
levels. Whether increased harvest indices can also be obtained for
other organs, such as tubers or seeds, remains to be determined.
In this paper we addressed the question of whether improvement of
photosynthetic performance by overexpressing phytochrome can be
realized without causing the adverse effects described above. For this
purpose, Arabidopsis phyB was used. Arabidopsis plants overexpressing
either Arabidopsis or rice phyB have been reported to show a
light-exaggerated phenotype, with reduced hypocotyl length and
increased chlorophyll content (Wagner et al., 1991; McCormac et al.,
1992; Wester et al., 1994). Tobacco plants overexpressing Arabidopsis
phyB also show semidwarfism (Halliday et al., 1997) but, to our
knowledge, detailed physiological studies have not yet been reported.
Here we describe the morphological and physiological characterization
of transgenic potato (Solanum tuberosum) plants grown under
white light in the greenhouse. By using potato as a host, we were able
to test the hypothesis that enhancing the responsiveness to white light
results in higher yields of storage organs.
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MATERIALS AND METHODS |
Plant Material
Plasmid pMAB316 (Wagner et al., 1991) encodes the Arabidopsis
PHYB cDNA under the control of the CaMV 35S promoter.
Transformation of potato (Solanum tuberosum cv
Désirée) plants with pMAB316 was performed according to the
method of Rocha-Sosa et al. (1989). We selected two lines with
different expression levels on the basis of reduced internode
elongation in white light. These lines were named "Dara" after the
host cultivar (Désirée) and the donor species
(Arabidopsis).
Growth Conditions
Plants cultivated in a sterile culture (Murashige and Skoog, 1962)
in a controlled-environment chamber (CU 32-L, Percival Scientific,
Boone, IA) at photon flux densities of approximately 0.1 mmol
m 2 s1 white light and
at 24°C (day)/22°C (night) were transferred to soil (maximum pot
diameter, 20 cm) and grown in a greenhouse at 0.15 to 0.5 mmol
m2 s1 white light,
15°C to 35°C, and a RH of approximately 55%. After 2 weeks, all
but the two strongest shoots were cut to obtain plants of comparable
strength.
RNA Extraction and Northern Analysis
Preparation of poly(A+) RNA from leaves and
subsequent northern analysis were performed as described by Heyer and
Gatz (1992a). Arabidopsis phyB transcripts were probed with
a 3.9-kb KpnI fragment generated from pMAB316 and potato
phyB transcripts with a 2.7-kb HpaI/KpnI fragment of a plasmid encoding the
full-length cDNA (Ruddat et al., 1997). Probes against the potato
ribosomal protein S4 (Devi et al., 1989) served to normalize for equal
loading of poly(A+) RNA. Radioactive signals were
detected with a bio-imaging analyzer (BAS 1000, Fuji, Tokyo) and
quantified with TINA 2.0 software (Raytest, Straubenhardt, Germany).
Protein Extraction and Immunoblot Analysis
Protein extraction and immunoblotting were performed as described
by van Tuinen et al. (1995) with slight modifications. The protein
pellet (corresponding to 0.3 g of leaf material) resulting from
the ammonium sulfate precipitation was resuspended in 15 µL of 0.5 M Tris buffer, pH 6.8. The protein concentration of the
extract was determined (Bradford, 1976) and the extract was dissolved
at 100°C for 2 min in 4× sample buffer (Laemmli, 1970). Equal
amounts of protein (100 µg) were separated on a 7% SDS-PAGE gel and
electroblotted onto a PVDF membrane (Millipore). Poinceau staining
confirmed uniform protein loading and homogenous blotting. The membrane
was incubated with either a mixture of three monoclonal antibodies
raised against Arabidopsis phyB in a mouse (1:5000 dilution; Somers et
al., 1991) or a polyclonal serum raised against potato phyB in a rabbit
(1:400 dilution). After treatment with peroxidase-conjugated secondary
antibody (1:1000 dilution, anti-mouse or anti-rabbit; Amersham), the
enhanced chemiluminescence kit (Amersham) visualized phyB and Aida 2.0 software (Raytest) quantified it densitometrically. Separate tests
confirmed that the phyB signal was proportional to the amount of
protein loaded.
Phenotypic Analysis
Stem height was measured as the distance from the apex to the soil
surface. For determination of the leaf-to-stem-weight ratio, representative plants were cut above the soil and the leaf laminas were
separated from the petioles and stems. Stem circumferences were
measured 1 cm above the soil. Weighing leaf discs randomly cut from
mature leaves determined the specific leaf fresh weight (milligrams per
centimeter). We estimated total leaf area (centimeters square) per
plant from the mean specific leaf weight and the total leaf weight per
plant. We determined tuber yield by measuring tuber number, total tuber
fresh weight per plant, and fresh weight per single tuber, considering
only tubers of at least 1 g. The tubers were harvested after
growth for approximately 5 months in two consecutive winters (until
February 1998 and April 1997). In the summer plants aged much earlier,
living only to a maximum age of 3 months, due to higher temperatures
and stronger pathogen exposure. In addition, developing tubers that had
grown during summer and winter were harvested from a few green plants
at 3 months.
Chlorophyll Determination
Leaf discs (1.3 cm2) were ground in a mortar
with liquid nitrogen; the chlorophyll was quantitatively extracted with
80% acetone in the presence of approximately 1 mg of
NaCO3. After the sample was centrifuged for 2 min
at maximum speed, we determined the total chlorophyll content
(chlorophyll a and b) of the supernatant photometrically (Uvicon 932, Kontron, Neuzahrn, Germany), according to
the method of Lichtenthaler (1987). Chlorophyll contents were in terms
of leaf area or leaf fresh weight.
Photosynthesis Measurements
We determined the CO2 uptake per leaf area
using IR spectroscopy with a transportable gas-exchange porometer (ADC,
Hoddesdon, UK), consisting of a central LCA-3 analyzer and a PLC-3 leaf
chamber for simultaneous recording of photon fluxes and endogenous
chamber temperature. An integrated personal computer stored and
processed the data. The terminal leaflets of leaves 6 to 8 (leaf 1 was
the first leaf larger than 1 cm) of 32- to 37-d-old plants were used. Before measurement, the leaflet was fixed in the chamber and
exposed to 50 to 500 µmol m2
s1 white light provided by a 150-lux lamp
(Flexilux, Schölly Fiberoptik, Denzlingen, Germany) at 22°C to
25°C until CO2 assimilation reached a maximum
steady-state level (10-15 min).
High-Light Studies
To study light-stress sensitivity, we exposed attached leaves
(leaves 6-8 of 32- to 37-d-old plants) for 5 h to 1.8 mmol
m2 s1 white light
(Power Star HQIT N/E, 2000 W, Osram, Munich, Germany) at a maximum (fan
cooled) leaf temperature of 38°C. We again determined CO2-assimilation rates as described above.
Additionally, leaf discs from three different plants were floated on
water and exposed to the same light source at 25°C to 30°C for
5 h. The ratio of variable to maximum chlorophyll a
fluorescence recorded with a fluorometer (PAM 101, Walz, Effeltrich,
Germany; kindly provided by Dr. K. Raschke, Göttingen), after 10 min of dark adaptation, served as a measure of photosynthetic
efficiency (Krause and Weis, 1991). High-light phenotypes were
investigated by cultivating plants under the same light source between
1.5 and 1.8 mmol m2 s1
white light (continuously fan cooled).
Preparation and Documentation of Light Micrographs
Cross-sections were cut from the center of mature, nonsenescent
leaf laminas of 6-week-old plants. The preparation procedure followed
the paraffin method (Gerlach, 1969). A microscope (DNLS, Leica)
magnified and a camera (NPS48, Leica) photographed the cross-sections.
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RESULTS |
Determination of phyB Transcript and Protein Levels
Potato plants were transformed with the Arabidopsis
phyB cDNA under the control of the CaMV 35S promoter. After
regeneration of 12 independent transformants, we selected two plants
exhibiting different degrees of stem elongation suppression in white
light for further characterization. Northern analysis of
poly(A+) RNA from leaves (Fig.
1) showed that the potato line Dara-5 expressed 0.2 relative units (the phyB signal divided by the S4 signal), whereas Dara-12 expressed 3.9 relative units of phyB mRNA. No
signal was obtained in wild-type plants. Transcription of the
endogenous PHYB was not significantly affected in the
transgenic lines.
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| Figure 1.
Northern analysis showing Arabidopsis phyB
(A.th. PhyB) and potato PhyB (S.t. PhyB)
transcript levels in leaves of wild-type (Wt), Dara-5, and Dara-12
potato plants. Hybridization with a potato S4 probe was done to correct
the signal for loading differences; 1.5 µg of poly(A+)
RNA was loaded per lane.
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For determination of phyB protein levels, we used monoclonal antibodies
raised against Arabidopsis phyB (Fig. 2;
Somers et al., 1991) to perform immunoblots with protein extracts from
leaves. Because the applied antibodies recognized equally Arabidopsis and potato phyB, the signal obtained with wild-type extracts
represented the amount of endogenous potato phyB. Densitometric
quantification of three blots indicated approximately 4-fold (Dara-5)
and 20-fold (Dara-12) overexpression of phyB in the transgenic lines
compared with the wild type. Low-temperature fluorescence measurements (Sineshchekov et al., 1996) on etiolated sprouts served to prove spectral activity of the transgenic phytochrome. Dark-grown sprouts (lower stem part) yielded 1.80 ± 0.49 relative fluorescence units in the wild type and 2.46 ± 0.44 relative fluorescence units in the transgenic Dara-12 plants. Irradiation for 3.5 h with red light depleted the phytochrome pool in wild-type plants to levels below
0.1 relative units, due to selective degradation of phyA. In Dara-12
plants, 30% of the total phytochrome survived this light treatment,
yielding relative fluorescence units of 0.82 ± 0.08 and
indicating at least an 8-fold overexpression of phyB in the sprouts of
this transgenic line.
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| Figure 2.
Immunoblot showing expression levels of phyB in
leaves of wild-type (Wt), Dara-5, and Dara-12 potato plants using a
monoclonal antibody raised against Arabidopsis phyB. Both Arabidopsis
phyB and endogenous potato phyB were equally recognized by this
antibody; 100 µg of protein was loaded per lane.
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Shoot Phenotype
Phenotypic differences between the shoots of wild-type and
transgenic plants were not evident when plants were grown in sterile culture, but strong differences in internode length and leaf
pigmentation developed about 10 d after transfer to soil and
greenhouse conditions. Soil-grown transgenic plants exhibited several
features also observed in other phyA- or phyB-overexpressing plants:
Arabidopsis, tomato, and tobacco grown in white light (Fig.
3, a and b; Boylan and Quail, 1989, 1991;
Keller et al., 1989; Wagner et al., 1991; Halliday et al., 1997).
Internodes of the phyB overexpressors were much shorter but thicker,
leading to a semidwarf phenotype (37% of wild-type height) in the
moderately expressing Dara-5 and to a stronger dwarf phenotype (29% of
wild-type height) in the forcefully expressing Dara-12 (Table
I). Toward the end of the vegetative period (after flowering), these differences decreased substantially. Dara-12, however, never reached the height of wild-type plants. Apical
dominance was reduced, leading to increased stem branching (Fig. 3, a
and b). Biomass allocation was directed toward leaves, resulting in an
increased leaf-to-stem ratio and an increased amount of fresh leaf
material per plant (Table I).
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| Figure 3.
a, Phenotype of a representative wild-type (Wt)
potato. b, Phenotype of a 5-week-old Dara-12 potato plant showing
shortened internodes and increased branching. Dara-5, which moderately
expresses phyB, exhibits an intermediate phenotype (not shown; see data
in Table I). c, Phenotype of leaves of wild-type (Wt), Dara-5, and
Dara-12 plants grown under moderate light conditions (0.15-0.5 mmol
m2 s1) in a greenhouse. Leaves 5 (counted
from the apex) from 10-week-old plants are shown. Bar = 6 cm.
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Table I.
Anatomical characteristics of Dara-5 and Dara-12
shoots compared with wild-type potato plants
All measurements were done on 6- to 8-week-old plants.
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Leaf Characteristics
As shown in Figure 3c, leaves of the transgenic plants were
considerably smaller than wild-type leaves. However, the number of
leaves was higher, resulting in the same total leaf area per individual
plant (Table I). Increased total fresh weight per leaf area (14% for
Dara-5 and 23% for Dara-12, Table II)
resulted from the increased thickness of the leaves. Light micrographs of leaf cross-sections (Fig. 4) revealed
that the increased thickness was basically due to the increased length
of palisade cells in the leaf mesophyll (19% more for Dara-5 and 30%
more for Dara-12), whereas the dimensions of the spongy tissues
remained largely unaffected (Fig. 4; Table II). The enlarged palisade
cells contained more chloroplasts, resulting in strongly elevated
chlorophyll contents on a leaf-area basis (Fig.
5) but not on the basis of leaf fresh
weight (Table II). Despite the differences in chlorophyll content per
leaf area, fractional red-light absorbance (Dr. G.H. Krause,
Düsseldorf, Germany, personal communication) was very similar for
all three lines (0.79 relative unit for wild type and 0.80 relative
unit for the two transgenic plants). The impression of a darker
pigmentation was intensified by slightly higher amounts of anthocyanins
in the leaves (Fig. 3c), which increased considerably when the plants
grew under higher light intensities (see Fig. 7).
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| Figure 4.
Light micrographs of leaf cross-sections of
wild-type (a) and transgenic Dara-12 leaves (b). Representative
sections were taken from leaves 8 of 6-week-old plants. Bar = 150 µm.
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| Figure 5.
Mean chlorophyll content during the course of leaf
maturation and senescence of wild-type ( ), Dara-5 ( ), and Dara-12
( ) potato plants. The arrow indicates onset of flowering, which was
at about the same time for all lines. Day 1 was October 26, 1997. Each
point represents the mean chlorophyll content of 10 segments cut (two
were taken at d 11) in regular distances from leaflets of apical to
basal leaves. Data from two representative plants per line are
depicted. Because of the sampling, aging was slightly (about 3 weeks)
accelerated in all plants.
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| Figure 7.
Photosynthesis rates at 1.8 mmol photons
m2 s1 of wild-type, Dara-5, and Dara-12
before (0 h) and after high-light stress (under 1.8 mmol
m2 s1) for 5 h. Leaf material was the
same as for Figure 6. Data are from nine leaves of three plants from
each line (SD as indicated).
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Chlorophyll Pigmentation during the Course of Leaf Maturation and
Senescence
To study the course of maturation and senescence of the transgenic
plants, the mean chlorophyll contents were analyzed at different stages
of development. Leaf segments sampled at temporal intervals and at
regular distances from apical to basal leaflets were pooled, and
chlorophyll was extracted and analyzed photometrically. The results are
presented in Figure 5. The chlorophyll content per leaf area in the
transgenic leaves began to rise within the first 20 d after
transfer from sterile to soil culture. Soon after the chlorophyll
accumulation per leaf area reached a maximum, senescence started
simultaneously in both the transgenic and the wild-type leaves.
Flowering, which initiates senescence, started at approximately the
same time (d 47). The time needed for complete chlorophyll degradation,
however, extended the lifetime of photosynthetically active transgenic
plants by 3 to 4 weeks.
Photosynthetic Activity
Figure 6 presents the photosynthetic
activity of mature wild-type and transgenic leaves. Young leaves from
nonsenescent plants (32-37 d old, compare with Fig. 5) were used.
Photon fluxes of more than approximately 250 µmol
m2 s1 in the transgenic
plants showed higher rates of leaf-area photosynthesis than did those
in the wild-type plants (Fig. 6a). As the senescence-related breakdown
of chlorophylls proceeded (Fig. 5), the advantages in the
photosynthetic performance of the transgenic plants became even more
pronounced (data not shown). We observed no difference in
photosynthetic activity (Fig. 6b) when the chlorophyll content was
normalized. The increased rates per unit leaf area can thus be
attributed to thicker leaf cross-sections and elevated chlorophyll contents. Because the total amount of leaf area was unaffected, increases in photosynthetic rates per individual plant (23% and 30%
for Dara-5 and Dara-12, respectively) were extrapolated from the mean
rates of leaves 5 to 12.
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| Figure 6.
Photosynthesis rates of mature, nonsenescent
leaves (leaves 6-8) of 32- to 37-d-old wild-type, Dara-5, and Dara-12
plants. a, Rates of wild-type (), Dara-5 (), and Dara-12 () at
different photon fluxes (50-500 µmol m2
s1) normalized to leaf area (µmol CO2
m2 s1). Data points are means of
measurements of 4 to 15 leaves from eight plants of each line. b, Rates
at 500 µmol photons m2 s1 normalized to
chlorophyll contents at site of the investigated leaf (µmol
CO2 g1 s1). In a data points
are for 4 to 15 leaves from eight plants of each line; in b data
are from nine leaves of three plants of each line (SD = 0.5-2.6 µmol m2 s1 for Dara-5 or as
indicated). chl, Chlorophyll.
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No significant structural changes in the photosynthetic apparatus of
the transgenic leaves were detected. HPLC analysis of ethanolic leaf
extracts of 30- to 40-d-old plants (device kindly provided by Dr. P. Jahns, Düsseldorf; method modified from that of Gilmore and
Yamamoto, 1991) revealed no changes in the carotenoid composition (data
not shown). Also, the ratios of chlorophyll a to
b were almost identical in wild-type and transgenic leaves (2.6 ± 0.3 for wild type and Dara-5 and 2.7 ± 0.4 for
Dara-12). Finally, electron microscopy (kindly performed by Dr. S. Hillmer, Göttingen; data not shown) did not indicate substantial
changes or deformations in the structure of the chloroplasts of the
transgenic plants, as previously observed in oat phyA-overexpressing
tobacco plants (Sharkey et al., 1991).
Light-Stress Experiments
Because the transgenic leaves revealed a sun leaf phenotype, we
analyzed the susceptibility of the photosynthetic apparatus to strong
irradiance using the nonsenescent leaves of plants grown under moderate
white light (0.15-0.5 mmol m2
s1). After 5 h at 1.8 mmol
m2 s1 white light and
25°C to 30°C, maximum CO2 assimilation had
dropped to about 25% of that in the plants maintained under moderate
light in the wild type and to only 50% (Dara-5) and 60% (Dara-12) in the transgenics (Fig. 7). During that
period, the ratio of variable to maximum chlorophyll a
fluorescence, used as a measure of photosynthetic efficiency, declined
from approximately 0.8 in the dark controls to approximately 0.2 in the
wild-type leaves to 0.3 in the Dara-5 leaves and to 0.35 in the Dara-12
leaves. Both studies indicated a significantly lower sensitivity of the
transgenic leaves to photoinhibition.
To study phenotypes under high irradiation, five plants per line were
cultivated under artificial white light at 1.5 to 1.8 mmol
m2 s1 in a greenhouse.
Differences in internode length and leaf size were less pronounced
under these conditions. However, the differences in leaf thickness and
chlorophyll contents remained. The formation of anthocyanins in leaves
(primarily in the lower epidermis, near veins, and in the petiole and
rachis) and, to a smaller degree, also in stems (outer cortex cells) of
the transgenic plants was strongly enhanced compared with plants grown
under moderate light (compare Fig. 3c with Fig.
8).
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| Figure 8.
Leaf phenotypes of wild-type, Dara-5, and Dara-12
plants grown under high intensities of white light (1.5-1.8 mmol
m2 s1) in a greenhouse, demonstrating
increased anthocyanin contents. Leaves 5 of 8-week-old plants are
shown. Bar = 3 cm.
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Tuber Yields and Underground Organ Growth
The phenotypes of the underground organs were determined when the
plants were totally senescent after approximately 5 months of growth in
a greenhouse. Table III summarizes the
yield data and Figure 9 shows
representative underground organs of each line. Biomass allocation to
underground organs (tubers and the root and underground shoot systems)
increased in the transgenic lines. The number of tubers per plant was
approximately 2-fold higher in Dara-5 and 3-fold higher in Dara-12
compared with the wild type. The tuber weight per plant in Dara-5 and
Dara-12 increased by 56% and 30%, respectively, of the tuber weight
of the wild type; and the average tuber size decreased to 65% and
43%, respectively, of the tuber size of the wild type. Relative dry
weight and starch contents of the tubers (Dr. B. Marty, Golm, Germany,
personal communication) were constant in the wild type, Dara-5, and
Dara-12 (22 ± 1 and 15 ± 1, respectively; n = 15). However, when harvested before 3 months (repeated several times
on a small scale), no significant increases in organ expansion appeared
and depressed tuber yields were even found in the transgenic
lines. Table III shows the tuber weights of three representative
plants per line, harvested at 3 months. At that age, yields were about
14% (Dara-5) and 23% (Dara-12) lower in the transgenics compared with
the wild type.
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Table III.
Tuber yield of Dara-5, Dara-12, and wild-type
potato plants after decay of the shoots following growth for 5 months
and after only 3 months
Only tubers >1 g were considered.
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| Figure 9.
Expansion of underground organs and tuber yields
of the wild type (Wt), Dara-5, and Dara-12 harvested upon decay of the
aerial parts after 5 months of cultivation in a greenhouse. The
transgenic lines showed enlarged underground organs and more but
smaller tubers, resulting in increased total tuber weight per plant
(Table III). Bar = 5 cm.
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DISCUSSION |
Transgenic plants overexpressing different members of the
phytochrome family have been extremely valuable for elucidating the
specific functions of individual phytochromes (McCormac et al., 1992,
1993). Moreover, they have also been used to map functional domains
important for photosensory specificity, dimerization, signal
transduction, and light-dependent degradation (for review, see Quail,
1997). Because of suppression of the shade-avoidance response,
transgenic tobacco plants overexpressing oat phyA revealed an increased
leaf-harvest index, opening new avenues for biotechnological applications of transgene technology (Robson et al., 1996). Being particularly interested in the latter application, we generated transgenic potato plants overexpressing phyB. Potato was chosen as a
host to explore the possibility that improved harvest index could also
be applied to tubers.
By choosing phyB we tried to circumvent the adverse effects of phyA on
chloroplast structure (Sharkey et al., 1991). We preferred Arabidopsis
phyB over potato phyB because it encodes an N-terminal hydrophobic
extension that is missing in potato phyB (Heyer and Gatz, 1992b).
Deletion of this extension decreased the sensitivity of the protein to
red light (Wagner et al., 1996). Thus, we speculated that expression of
Arabidopsis phyB might be more effective than expression of potato
phyB. Transgenic potato plants transformed with the Arabidopsis phyB
cDNA under the control of the CaMV 35S promoter supported the notion
that phytochrome-overexpressing plants may have agricultural
importance. We discuss the data presented here in the context of
results previously obtained with other phyB- or phyA-overexpressing
plants.
Transgenic potato plants overexpressing Arabidopsis phyB exhibited a
semidwarf phenotype, with shorter and thicker stems, reduced apical
dominance, smaller but thicker leaves, a higher leaf-to-stem weight
ratio, increased chlorophyll content, higher rates of photosynthesis,
prolonged timespan for photosynthesis, and, when harvested after the
natural decay of the shoots, higher tuber numbers and yields.
Physiological data from other Arabidopsis phyB-overexpressing plants
under white light are limited. Transgenic Arabidopsis plants also
revealed an increased chlorophyll content. In addition, a slightly
reduced ratio of chlorophyll a to b was detected
(Wester et al., 1994), a phenomenon not observed in transgenic potato
plants. Like the transgenic potato plants described here, transgenic
tobacco plants overexpressing phyB showed reduced stem extension
(Halliday et al., 1997).
Transgenic tobacco plants overexpressing oat phyA have been studied in
more detail. Like phyB-overexpressing potato plants, phyA-overexpressing tobacco plants are characterized by semidwarfism, reduced apical dominance, smaller but thicker leaves, increased chlorophyll content, and delayed senescence (Keller et al., 1989; Cherry et al., 1991). The elicitation of this "light-exaggerated" phenotype by overexpression of either phyA or phyB is consistent with
the notion that a variety of developmental processes can be controlled
by either phytochrome, with phyA being activated by FR light and phyB
by red light (Quail et al., 1995). As white light contains red and FR
light, both types of overexpressors contain elevated levels of Pfr
under white light. The increased sensitivity of phyA-overexpressing
plants to FR light has been well documented (McCormac et al., 1992;
Robson et al., 1996). However, it remains to be shown that it is the
redlight component of white light that is responsible for the
light-exaggerated phenotype in the transgenic plants described here.
In terms of photosynthetic performance, phyB-overexpressing potato
plants were superior to phyA overexpressors (Sharkey et al., 1991).
Both types of phytochrome overexpressors had thicker leaves and higher
chlorophyll content per leaf area. Light micrographs illustrate that
leaf thickening was due to general enlargement of mesophyll cells in
phyA-expressing tobacco (Sharkey et al., 1991), whereas only palisade
cells were elongated in phyB-overexpressing potato (Fig. 4; Table II).
Electron micrographs from transgenic phyA overexpressors showed that
many of the chloroplasts were cup-shaped, with the middle portions of
the chloroplasts bowing away from the plasmalemma (Sharkey et al.,
1991). These abnormalities worsened the photosynthetic performance of
the plants at normal CO2 concentrations.
Abnormalities of the chloroplasts were not detectable in transgenic
phyB-overexpressing potato plants. In contrast, these plants
displayed higher photosynthesis rates (Fig. 6a). This effect was
proportional to the increased chlorophyll levels (Fig. 6b). The
differences in photosynthetic activity were even more pronounced in
senescing leaves as the relative differences in pigmentation increased.
In addition, photosynthesis was less sensitive to photoinhibition (Fig.
7), which may have been due to the higher chlorophyll content per unit
leaf area resulting in lower excitation of pigments compared with
wild-type leaves. Whether the stimulated anthocyanin formation observed
under high-light conditions was also involved in protecting the
transgenic photosynthesis apparatus (especially under high UV radiation
in the field) remains to be elucidated.
The enhanced responsiveness of the transgenics to white light led not
only to increased thickening of leaves, and thus to higher
photosynthetic performance and higher biomass production of the aerial
parts of the plants, but also to a preferential allocation of
assimilates to the leaves at the expense of the stems (Table I). This
is consistent with the phenotype of transgenic tobacco plants
overexpressing phyA (Robson et al., 1996). phyA-overexpressing plants
also displayed this altered assimilate allocation but, in contrast to
plants overexpressing phyB, only in response to low relative ratios of
red to FR light.
Both phyA-overexpressing tobacco plants and phyB-overexpressing potato
plants stayed green longer. To analyze whether this apparently delayed
senescence was due to a delayed beginning or to a slower senescing
process, we monitored the chlorophyll contents over the life cycle of
the phyB-overexpressing potato plants (Fig. 5). Chlorophyll levels of
wild-type and transgenic plants increased at different rates, resulting
in 35% more chlorophyll in the mature leaves of the transgenic lines.
Chlorophyll contents started to decrease at approximately d 40 in all
of the plant lines, indicating no difference in the start of
senescence. Also, the slope of the decline was almost identical.
However, assuming a similar amount of chlorophyll per chloroplast in
the nonsenescent state (deduced from the concomitant increases in
chloroplast number per cell and chlorophyll level per leaf area), the
rate of chlorophyll loss per individual chloroplast decelerated; the
time needed for complete chlorophyll degradation was several weeks
longer in the transgenic plants. This result is consistent with
findings of Cherry et al. (1991), who also observed a simultaneous
start but a decelerated senescence process in phyA-overexpressing
plants.
Tuber yield is the essential parameter for estimating the agricultural
potential of transgenic potato plants. Keiller and Smith (1989) have
shown that increasing the ratio of red to FR light for radish results
in preferential assimilate allocation to the storage organ at the
expense of leaf petiole extension. To our knowledge, similar effects
have not been reported in potato. By increasing phyB levels, and thus
presumably the sensitivity to red light, we explored the possibility of
stimulating photosynthesis and assimilating allocation to tubers using
one central regulator. When harvested after 3 months, tuber yields were
lower than in wild-type plants. phyB apparently delays tuber formation,
although we observed no effect on flowering. The delayed tuber
formation in phyB overexpressors corresponds well with accelerated
tuber formation in phyB antisense plants (Jackson et al., 1996). In these plants tuber formation was induced even under long-day conditions using the short-day potato cv Andigena. Because the work presented here
was done with the potato cv Désirée, which is under
less-stringent photoperiodic control, these experiments are not
directly comparable. Once tuber induction was initiated, the high
photosynthetic performance of even senescing plants led to storage of
elevated amounts of biomass into tubers, resulting in higher yields. In
spite of higher yields, average tuber size was smaller than in
wild-type plants. It may be speculated that decreased apical dominance
of the underground shoot leads to increased stolon formation. Increased
biomass allocation was not confined to tubers but was also present in
other underground organs such as roots and shoots.
The results of this study support the idea that improving agricultural
performance by altering phyB levels is feasible. Higher photosynthetic
rates led to more biomass, which is allocated into leaves but also into
underground organs such as tubers. Whether these features are
maintained under field conditions remains to be determined. Judging
from our experiments here, we believe that these plants are potentially
more productive, especially in areas with high irradiation; therefore,
reduced photoinhibition becomes advantageous. As plants are able to
assimilate CO2 for a longer time because of
decelerated chlorophyll breakdown, they might be preferentially
valuable in areas with long growing seasons.
| |
FOOTNOTES |
1
This work was supported by the European
Communities BIOTECH program (grant no. BIO2 CT-930400), by a Department
of Energy grant (no. DE-FG03-87ER13742) to P.H.Q., and by a U.S.
Department of Agriculture grant (no. CRIS 5335-21000-010-00D) to P.H.Q.
*
Corresponding author; e-mail cgatz{at}gwdg.de; fax
49-551-397820
Received September 22, 1998;
accepted January 26, 1999.
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
FR, far-red.
phyA and phyB, phytochromes A and B, respectively.
| |
ACKNOWLEDGMENTS |
We are grateful to Dr. V. Sineshchekov (Moscow State
University), who provided the data concerning low-temperature
fluorescence. We also wish to thank Dr. E. Reiner-Drehwald and G. Weis
for support with light microscopy, Dr. S. Hillmer for carrying out the
electron microscopy, and Dr. H.W. Heldt for providing his gas-exchange porometer. We give special thanks to F. Glasenapp and U. Wedemeyer, the
greenhouse staff in Göttingen, for skillfully cultivating the
plants.
| |
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Abstract
Introduction