PHYSIOLOGIA PLANTARUM 118: 456–463. 2003
Printed in Denmark – all rights reserved
Copyright # Physiologia Plantarum 2003
ISSN 0031-9317
Chloroplast biogenesis by Arabidopsis seedlings is impaired in the
presence of exogenous glucose
Jennifer P. C. Toa, Wolf-Dieter Reiterb and Susan I. Gibsonc,*
a
Biology Department, University of North Carolina, Coker Hall, CB#3280, Chapel Hill, NC 27599–3280 USA
Department of Molecular and Cell Biology, University of Connecticut, Box U-125, 75 North Eagleville Road, Storrs,
CT 06269–3125 USA
c
Department of Plant Biology, University of Minnesota, 220 Biological Science Center, 1445 Gortner Ave., St. Paul,
MN 55108–1095 USA
*Corresponding author, e-mail: [email protected]
b
Received 2 April 2002; revised 21 December 2002
Seedlings of Arabidopsis thaliana (L.) Heynh. fail to
become green when germinated and grown on media
containing high concentrations of glucose (Glc). Although
previous studies have shown that sugar concentration
affects chlorophyll levels and photosynthetic gene expression, the possibility that sugar concentration might affect
actual chloroplast biogenesis has received little attention.
Therefore, experiments were conducted to determine whether
germination and growth on Glc impairs development of
mature chloroplasts from the proplastids found in plant
embryos. To monitor chloroplast biogenesis, the levels of a
chloroplast-specific fatty acid, hexadecatrienoic (16:3) fatty
acid, were measured in Arabidopsis seedlings grown on media
containing different concentrations of Glc. These experiments
indicate that moderate concentrations of Glc delay accumulation
of 16:3. The effects of Glc on 16:3 levels are not solely due to
osmotic stress, as equi-molar and even twice equi-molar
concentrations of sorbitol do not exert comparable effects.
Seedlings grown on concentrations of Glc high enough to
prevent greening accumulate almost no 16:3, even after
22 days of growth under continuous light conditions. The
lack of 16:3, a major structural component of chloroplast
membranes, suggests that seedlings do not develop mature
chloroplasts when grown in the presence of high concentrations
of exogenous Glc. Further support for this hypothesis is
provided by electron microscopy studies revealing that seedlings grown on high concentrations of Glc lack identifiable
chloroplasts. Although Glc has been reported to inhibit chloroplast development in unicellular organisms, similar studies on
intact higher plants have been lacking.
Introduction
In addition to playing a central role in metabolism,
soluble sugars also act as signalling molecules. For
example, the phenomenon of carbon catabolite repression,
wherein glucose (Glc) represses expression of specific
genes, has been well documented in bacteria (Ullmann
1996) and yeast (Gancedo 1998). In contrast, the effects
of sugar levels on other cellular processes have been less
well characterized. For instance, little is known about the
effects of soluble sugar levels on organelle biogenesis.
The majority of the few studies to date on the effects of
soluble sugar levels on organelle biogenesis have focused
on unicellular organisms. Results from these studies indi-
cate that soluble sugars, such as Glc, typically inhibit
organelle biogenesis. For example, increasing Glc levels
decreases the ratio of mitochondrial DNA to nuclear
DNA in the yeast Saccharomyces carlsbergensis. In addition, increasing Glc levels causes a decrease in the average
number of mitochondria per cell (Bleeg et al. 1972). Similarly, an increase in Glc levels leads to a decrease in the ratio
of mitochondrial to nuclear DNA and protein synthesis in
Saccharomyces cerevisiae (Böker-Schmitt et al. 1982).
Increasing Glc levels have also been shown to inhibit
chloroplast biogenesis or promote chloroplast degeneration in unicellular organisms. In the unicellular green
Abbreviations – 16:0, palmitic fatty acid; 16:3, hexadecatrienoic fatty acid; 18:2, linoleic fatty acid; 18:3, linolenic fatty acid; 20:1, eicosenoic
fatty acid; Glc, glucose; Sorb, sorbitol; Suc, sucrose.
456
Physiol. Plant. 118, 2003
algae Chlorella protothecoides, increasing the concentration of Glc in the growth media leads to a decrease in the
production of sulfolipids that are found predominantly in
chloroplast membranes (Shibuya and Hase 1965). More
direct evidence for Glc-mediated inhibition of chloroplast
biogenesis in Chlorella comes from electron microscopy
studies. These studies show that transferring Chlorella to
high-Glc media results in chloroplast degeneration. Upon
transfer of cells to high-Glc media, the chloroplasts expand
and their lamellae become diffuse (Shihira-Ishikawa and
Hase 1964). Furthermore, cells grown in the presence of
high concentrations of Glc eventually bleach, losing their
green pigmentation. Electron microscopy studies of ‘Glcbleached’ Chlorella indicate that they lack identifiable
chloroplasts (Oh-Hama et al. 1965). Similar studies on
the unicellular flagellate Euglena gracilis indicate that
addition of Glc to the growth media inhibits chloroplast
lengthening and the rate of thylakoid formation and
fusion (Schwelitz et al. 1978). Glc-mediated inhibition of
chloroplast development has been postulated to be
advantageous to cells, as it reduces synthesis of chloroplast materials under conditions where there is less need
to produce sugars through photosynthesis (Kirk 1967).
Sugars also help regulate many physiological and
developmental processes in higher plants (Thomas and
Rodriguez 1994, Graham 1996, Koch 1996, Jang and
Sheen 1997, Smeekens and Rook 1997, Smeekens 1998,
Gibson and Graham 1999, Sheen et al. 1999, Yu 1999,
Gibson 2000, Pego et al. 2000, Smeekens 2000, Coruzzi
and Bush 2001, Coruzzi and Zhou 2001, Rolland et al.
2002). In higher plants, sugar levels have been shown to
affect chlorophyll accumulation and the expression of
several genes involved in photosynthesis (Posner 1970,
Sheen 1990, Krapp et al. 1991, Krapp et al. 1993, Jang
and Sheen 1994, Knight and Gray 1994, Sheen 1994, van
Oosten et al. 1994, Dijkwel et al. 1996, Dijkwel et al.
1997). However, these studies did not directly examine
the effects of sugars on chloroplast development. Although
sucrose (Suc) has been shown to cause a reduction in
chloroplast number in carrot callus cultures (Edelman
and Hanson 1971), similar studies on the effects of sugars
on chloroplast development in intact, developing plants
have been lacking. Here we report that seedlings of
Arabidopsis thaliana grown in the presence of high
concentrations of Glc fail to produce significant quantities of a fatty acid, hexadecatrienoic (16:3) fatty acid,
that is a major structural component of chloroplast
membranes. In addition, electron microscopy studies
reveal that these plants lack identifiable chloroplasts.
Materials and methods
Materials and growth conditions
Wild-type Arabidopsis thaliana var. Columbia seeds were
originally obtained from Dr Chris Somerville (Carnegie
Institution of Washington, Palo Alto, CA, USA). Seeds
were sown on 3MW Gel Blot Paper (Midwest Scientific,
Valley Park, MO, USA) on Petri plates containing solid
Physiol. Plant. 118, 2003
Arabidopsis minimal media (Kranz and Kirchheim 1987)
supplemented with the indicated additives. Plants were
grown at 21–25 C, under 60–80 mmol photons m2 s1
continuous fluorescent light.
Transmission electron microscopy
Cotyledons were prefixed by submersion under a vacuum
in 2.5% glutaraldehyde, 0.1 M sodium phosphate
(pH 7.0) for 5 h. Specimens were washed with 0.1 M
sodium phosphate (pH 7.0) and then post-fixed in 1%
osmium tetroxide, 0.1 M sodium phosphate (pH 7.0) for
1 h in the dark. Both fixation steps were performed at
room temperature. Fixed tissues were washed with 0.1 M
sodium phosphate (pH 7.0) prior to in-block staining
with 1% uranyl acetate overnight at 4 C in the dark.
Stained tissues were washed with water and dehydrated
over a graded ethanol series (Roland and Vian 1991).
Dehydrated tissues were transferred to propylene oxide
and embedded in Eponate 12 medium (Ted Pella Inc.,
Redding, CA, USA). The medium was allowed to harden
at 60 C for 2 days. Embedded tissue blocks were precision
trimmed and ultra-thin sectioned on a microtome. Sections
were collected on copper grids and stained with lead citrate
for 10 min to improve contrast. After washing and drying,
the sections were visualized and photographed using a
JEOL 2010 transmission electron microscope (JEOL USA
Inc., Peabody, MA, USA), operated at 80 kV at a magnification of 4000.
Fatty acid extraction and gas chromatography
Lipid extractions and fatty acid derivatizations to the
corresponding methyl esters were performed using an
established procedure (Browse et al. 1986). Approximately
25–50 seeds/seedlings were used for each assay. Seeds/
seedlings were incubated for 1–2 h in 0.6–2.0 ml 1 N
methanolic-HCl (Supelco, Bellefonte, PA, USA) at
80–83 C. The fatty acid methyl esters were then extracted
into hexane and analysed by gas chromatography as
described previously (Chia et al. 2000).
Fatty acid identification and quantification
Gas chromatograph peaks were identified and quantified
by comparing their sizes and column retention times with
the sizes and retention times of fatty acid methyl ester
standards (Sigma, St. Louis, MO, USA). Using this data,
the net amounts of membrane fatty acids generated by the
seedlings in each sample were calculated as previously
described (To et al. 2002). In brief, the percentage of seed
storage fatty acid remaining at day ‘x’ was calculated
based on the percentage of eicosenoic (20:1) fatty acid
remaining at day ‘x’. Eicosenoic fatty acid comprises
17% of seed storage fatty acid but is present in only
trace amounts in membrane fatty acids (Lemieux et al.
1990). Therefore, decreases in eicosenoic fatty acid levels
reflect decreases in seed storage lipid levels. The absolute
amounts of seed storage fatty acid remaining at day ‘x’
457
were calculated based on findings that, in oilseed plants,
the vast majority of seed fatty acids are present in storage
lipids. For example storage lipids represent 93% of the
total lipid present in seeds of Brassica napus, a species
closely related to Arabidopsis (Norton and Harris 1975).
The net increases in membrane fatty acids at day ‘x’ were
then calculated by subtracting the amounts of membrane
fatty acids present in seeds and the amounts of storage
fatty acids remaining at day ‘x’ from the total amounts
of fatty acids present at day ‘x’.
Media-shift experiments
Seeds were sown on nytex mesh 3–300/46 screens (Tetko
Incorp. Kansas City, MO, USA) placed on 3MW Gel
Blot Paper on Petri plates containing solid media. At the
indicated times, the nytex screens and seeds/seedlings
were shifted to Petri plates containing fresh solid
media, covered with 3MW Gel Blot paper.
Results
Production of chloroplast membrane lipids is retarded in
the presence of exogenous Glc
Arabidopsis thaliana seedlings grown in the presence of
high concentrations (e.g. 0.27–0.33 M) of exogenous Glc
or Suc develop very little green pigmentation (Jang et al.
1997, Németh et al. 1998, Zhou et al. 1998, Arenas-Huertero
et al. 2000, Huijser et al. 2000, Laby et al. 2000, Gibson et al.
2001). As shown in Fig. 1, seedlings grown on media
containing 0.3 M Glc have purple cotyledons and very pale
green hypocotyls. In contrast, seedlings grown on equimolar concentrations of sorbitol or on low (e.g. 0.03 M)
concentrations of Glc develop dark green shoot systems
(Fig. 1). A possible explanation for these findings is that
the development of mature chloroplasts from the proplastids
found in plant embryos might be impaired in the presence
of moderate to high concentrations of exogenous Glc.
To determine whether chloroplast formation is
inhibited by Glc, the levels of a chloroplast-specific fatty
acid, hexadecatrienoic (16:3) fatty acid, were measured in
seedlings grown on different concentrations of Glc. Hexadecatrienoic fatty acid constitutes a major component of
chloroplast membranes, but is almost entirely lacking
from other membranes and from seed storage lipids
(Harwood 1980, Lemieux et al. 1990). As a result, synthesis of chloroplast membranes, and therefore of chloroplasts, can be monitored by measuring 16:3 levels. As
shown in Fig. 2, seedlings grown on media containing
0.14 M Glc exhibit a significant decrease in the rate of
16:3 accumulation relative to seedlings grown on 0.03 M
Glc. This result suggests that formation of mature chloroplasts by developing seedlings is impaired in the presence
of moderate (0.14 M) concentrations of Glc.
Although seedlings grown on 0.27 M sorbitol 1 0.03 M
Glc develop green shoot systems, seedlings grown on
somewhat higher concentrations of sorbitol (e.g. 0.4 M
sorbitol 1 0.03 M Glc) become only pale green (Fig. 1).
458
Fig. 1. Seedlings grown on high concentrations of exogenous sugar
fail to become green. Arabidopsis seedlings were grown under
continuous light for 21 days on minimal Arabidopsis media
supplemented with the indicated additives. Red bars ¼ 2.0 mm.
Glc, glucose; Sorb, sorbitol.
This finding raised the possibility that the effect of
Glc on chloroplast development is due to osmotic
stress. To determine whether Glc affects chloroplastspecific fatty acid production via alterations in the
osmotic potential of the growth media, the effect of sorbitol on 16:3 levels was also examined. As shown in
Fig. 2, growth on equi-molar and almost twice equimolar concentrations of sorbitol has less effect on 16:3
levels than growth on 0.14 M Glc. These results indicate
that osmotic stress is not sufficient to explain the effects
of Glc on 16:3 production.
Seedlings grown on high concentrations of exogenous Glc
fail to produce identifiable chloroplasts or significant
quantities of chloroplast membrane lipids
The finding that moderate (e.g. 0.14 M) concentrations
of Glc retard accumulation of chloroplast-specific fatty
Physiol. Plant. 118, 2003
0.03 M Glc
0.14 M Gl c
0.11 M Sorb + 0.03 M Glc
0.22 M Sorb + 0.03 M Glc
16:3 Fatty Acid (µg/Seed)
0.14
0.12
0.10
0.08
0.06
0.04
0.02
8
6
4
2
0
0.00
Days
Fig. 2. Accumulation of chloroplast-specific fatty acid is retarded in
the presence of exogenous Glc. Hexadecatrienoic (16:3) fatty acid
levels were measured in Arabidopsis seeds/seedlings harvested from
the indicated media at different times after the start of imbibition.
Results presented are means – SD (n ¼ 3). Note that in some cases the
error bars are smaller than the plot symbols and so are not visible.
This experiment was repeated, with similar results. Glc, glucose; Sorb,
sorbitol.
acids (Fig. 2) raises the possibility that seedlings grown
on higher concentrations of Glc might be unable to form
mature chloroplasts. To examine this possibility, transmission electron microscopy was used to visualize cotyledon cells from seedlings grown on high (0.3 M) or low
(0.03 M) Glc. As shown in Fig. 3, cells from seedlings
grown on low Glc contain numerous chloroplasts. In
contrast, cells from seedlings grown on high Glc contain
no structures with the thylakoid membrane systems characteristic of mature chloroplasts. Instead, these cells
undergo little expansion and are packed with oil bodies
and other structures (Fig. 3). To obtain more quantitative
data, 16:3 levels were also measured in seedlings grown
on high or low Glc. As shown in Fig. 4, plants grown on
0.3 M Glc contain only trace amounts of 16:3, even after
22 days of growth under continuous light conditions. In
comparison, plants grown on 0.03 M Glc accumulate
approximately 30 times as much 16:3 as plants grown
on 0.3 M Glc (Fig. 4).
Specificity of the effect of exogenous Glc on 16:3
accumulation
High concentrations of Glc severely inhibit seedling
growth (Fig. 1). Therefore, it was of interest to determine
whether the effects of Glc on 16:3 accumulation are
relatively specific, or are simply a consequence of an
overall reduction in seedling growth. Towards this end,
the level of each major fatty acid species was measured in
seedlings grown on different media. This data was used
Physiol. Plant. 118, 2003
to calculate net accumulation of each major fatty acid in
the membranes of a given group of seedlings. Four fatty
acids, palmitic (16:0), hexadecatrienoic (16:3), linoleic
(18:2) and linolenic (18:3), together account for approximately 90% of the membrane fatty acids present in each
group of seedlings (Fig. 4). Unlike 16:3 fatty acid, 16:0,
18:2 and 18:3 fatty acids are abundant in non-chloroplast
membranes as well as being present in chloroplast membranes (Harwood 1980). Therefore, if high Glc inhibits
the formation of chloroplast membranes more than the
formation of other membranes, 16:3 accumulation
should be inhibited to a greater extent than accumulation
of 16:0, 18:2 and 18:3.
As shown in Fig. 4, seedlings grown on high (0.3 M)
Glc accumulate significantly smaller quantities of all four
major fatty acids than seedlings grown on equi-molar
sorbitol or on lower concentrations of Glc. However,
the effects of high Glc on 16:3 accumulation are particularly severe. Accumulation of 16:3 accounts for approximately 6.8% of total membrane fatty acid accumulation
by seedlings growing on 0.03 M Glc or 0.27 M sorbitol 1
0.03 M Glc, but only about 1.5% of total membrane
fatty acid accumulation by seedlings grown on 0.3 M
Glc (Fig. 4). In contrast, the relative levels of accumulation of 16:0, 18:2 and 18:3 in membranes are almost
identical for seedlings grown on 0.03 M Glc, 0.3 M Glc
or 0.27 M sorbitol 1 0.03 M Glc. For example, 18:2 accumulation accounts for approximately 20% of the increase
in total membrane fatty acids in seedlings grown on all
three media.
Although seedlings grown on 0.27 M sorbitol 1 0.03 M
Glc accumulate each of the four most abundant fatty
acids in the same ratios as seedlings grown on 0.03 M Glc
(Fig. 4), growth on higher concentrations of sorbitol (e.g.
0.4 M sorbitol 1 0.03 M Glc) does cause alterations in
the relative increases in the amounts of different membrane fatty acids. Accumulation of both 16:3 and 18:2
comprises a lower percentage of total membrane fatty
acid accumulation by seedlings grown on 0.4 M sorbitol
1 0.03 M Glc than by seedlings grown on 0.03 M Glc.
Conversely, 18:3 accumulation represents a greater percentage of total membrane fatty acid accumulation by
seedlings grown on 0.4 M sorbitol 1 0.03 M Glc than by
seedlings grown on 0.03 M Glc (Fig. 4). So, although
growth on both high (0.3 M) Glc and high (0.4 M) sorbitol
inhibits fatty acid accumulation, high Glc has a particularly severe effect on 16:3 accumulation whereas high
sorbitol alters the relative amounts of several fatty
acids. These results suggest that sorbitol affects overall
fatty acid composition rather than affecting primarily
chloroplast-specific fatty acids.
Seedlings lose susceptibility to the inhibitory effects of
exogenous Glc on chloroplast membrane formation within
3 days of the start of imbibition
Previous work has shown that seedlings become insensitive to the inhibitory effects of high sugar concentrations
on early seedling development within 2–3 days of the
459
Fig. 3. Seedlings grown on
high concentrations of
exogenous Glc lack
identifiable chloroplasts.
Arabidopsis seedlings were
grown under continuous light
for 13 days on minimal
Arabidopsis media
supplemented with 0.03 M or
0.3 M Glc. Cotyledon sections
were visualized using a
transmission electron
microscope. Structures
containing the distinctive
thylakoid membrane systems
found in mature chloroplasts
could easily be identified in all
tissue sections from plants
grown on 0.03 M Glc but
could not be identified in any
of the tissue sections from
plants grown on 0.3 M Glc.
Figures depict representative
cells. Note that the upper-right
and bottom figures are
composites, formed by
combining separate
photographic images of
different portions of a single
cell. Arrows indicate positions
of some of the chloroplasts
visible in the cell from the
seedling grown on 0.03 M Glc.
The bars in the bottom right
corner of each figure ¼ 4.0 mm.
Glc, glucose.
start of imbibition (Gibson et al. 2001). To determine
whether seedlings also become resistant to the negative
effects of high Glc on chloroplast development, seedlings
were either sown directly on high Glc or were transferred
to high Glc after 3 days on low Glc. As shown in Fig. 5,
seedlings sown on low (0.03 M) Glc and then transferred
to high (0.3 M) Glc actually accumulate more total
membrane fatty acid, as well as more 16:3, than seedlings
grown continuously on low Glc. However, 16:3 accumulation comprises approximately the same percentage of
total membrane fatty acid accumulation for both sets of
seedlings. In contrast, 16:3 accumulation forms a relatively
small percentage of total membrane fatty acid accumulation by seedlings sown directly on high (0.3 M) Glc.
460
As an osmotic control, seedlings were sown directly on
0.27 M sorbitol 1 0.03 M Glc or were shifted to that
media after 3 days on 0.03 M Glc. As shown in Fig. 5,
both sets of seedlings exhibit reductions in 16:3 and total
membrane fatty acid accumulation. In contrast to 0.3 M
Glc, transferring seedlings to 0.27 M sorbitol 1 0.03 M
Glc does not result in substantially different levels of
16:3 or total membrane fatty acid accumulation than
sowing seeds directly on 0.27 M sorbitol 1 0.03 M Glc.
Furthermore, 16:3 represents about the same percentage
of total membrane fatty acid accumulation for seedlings
sown directly on 0.27 M sorbitol 1 0.03 M Glc or transferred to that media after 3 days on 0.03 M Glc as for
seedlings grown on 0.03 M Glc.
Physiol. Plant. 118, 2003
Total Membrane Fatty Acid
Accumulation (µg/Seed)
14
12
10
8
6
4
2
The effects of growth on 0.4 M sorbitol 1 0.03 M Glc
were also examined. This concentration of sorbitol
severely affects seedling development (Fig. 1). As shown in
Fig. 5, seedlings sown directly on 0.4 M sorbitol 1 0.03 M
Glc exhibit dramatic reductions in both total and
16:3 fatty acid accumulation. However, in contrast to
the effects of high (0.3 M) concentrations of Glc, much
of the inhibitory effect of 0.4 M sorbitol 1 0.03 M Glc on
total and 16:3 fatty acid accumulation remains 3 days
Physiol. Plant. 118, 2003
16:3 Fatty Acid Accumulation
(µg/Seed)
10
1.0
0.8
0.6
0.4
0.2
0.0
8
6
4
2
0.03 M Glc to
0.4 M Sorb + 0.03 M Glc
0.4 M Sorb + 0.03 M Glc
0.03 M Glc to
0.27 M Sorb + 0.03 M Glc
0.27 M Sorb + 0.03 M Glc
0.03 M Glc to 0.3 M Glc
0.3 M Glc
0
0.03 M Glc
Fig. 4. Accumulation of chloroplast-specific fatty acids is specifically inhibited by growth on high concentrations of Glc. Seedlings
were grown under continuous light on the indicated media for
22 days. The majority of seedlings grown on 0.3 M Glc or 0.4 M
sorbitol 1 0.03 M Glc exhibit arrested development. However, some
of the seedlings grown on these media escape the selection, as shown
by development of relatively normal shoot systems, and were not
collected for analysis. Seedlings grown on 0.03 M Glc or 0.27 M
sorbitol 1 0.03 M Glc exhibit a nearly uniform morphology, and so
all seedlings from these media were collected for analysis. The levels
of all major fatty acid species were determined for each sample. This
data was used to calculate the increases in the amounts of each of
the four most abundant fatty acids present in the seedling
membranes (top panel). See the ‘Materials and methods’ section
for a description of the method used to estimate accumulation of
membrane fatty acids. The percentage of total membrane fatty acid
accumulation comprised by each of the four most abundant
membrane fatty acids is also depicted (bottom panel). Results
presented are means – SD (n ¼ 3). Note that in some cases the error
bars are smaller than the plot symbols and so are not visible. This
experiment was repeated, with similar results. Glc, glucose; Sorb,
sorbitol.
1.2
16:3 Fatty Acid Accumulation
(% of Total Membrane Fatty Acid)
0
Fig. 5. Seedlings become resistant to the inhibitory effects of Glc on
chloroplast-specific fatty acid accumulation within 3 days of the
start of imbibition. Seeds were sown on minimal media supplemented with 0.03 M Glc, 0.3 M Glc, 0.27 M sorbitol 1 0.03 M Glc
or 0.4 M sorbitol 1 0.03 M Glc. After 3 days, some of the seeds/
seedlings sown on 0.03 M Glc were transferred to media
supplemented with 0.3 M Glc, 0.27 M sorbitol 1 0.03 M Glc or
0.4 M sorbitol 1 0.03 M Glc. The levels of all major fatty acid
species were measured in seedlings harvested after an additional 7
days of growth. This data was used to calculate the quantities of
total membrane fatty acids and of 16:3 that accumulated in each
group of seedlings. This figure presents the combined results of two
independent experiments. Results presented are means – SD (n ¼ 6).
Note that in some cases the error bars are too small to be depicted.
Glc, glucose; Sorb, sorbitol.
after the start of imbibition. Furthermore, although 16:3
represents a somewhat smaller percentage of total
461
membrane fatty acid accumulation in seedlings sown
directly on 0.4 M sorbitol 1 0.03 M Glc than in seedlings
sown on low (0.03 M) Glc, the percentage of total membrane fatty acid accumulation represented by 16:3 in
0.4 M sorbitol 1 0.03 M Glc-grown seedlings is greater
to a statistically significant (P-value , 0.001 by a
Student’s t-test) degree than in 0.3 M Glc-grown seedlings.
Discussion
Arabidopsis thaliana seedlings grown in the presence of
high concentrations of Glc fail to turn green. One model
for the basis of this phenomenon is that Glc might inhibit
the development of mature chloroplasts from the small
proplastids found in plant embryos. Precedent for such a
model is provided by studies demonstrating that sugars
inhibit organelle development in at least some unicellular
organisms. For example, Glc inhibits chloroplast development in Euglena gracilis (Schwelitz et al. 1978) and
Chlorella protothecoides (Shihira-Ishikawa and Hase
1964, Oh-Hama et al. 1965, Shibuya and Hase 1965)
and mitochondrial formation in Saccharomyces carlsbergensis (Bleeg et al. 1972) and Saccharomyces cerevisiae
(Böker-Schmitt et al. (1982). In addition, Suc has been
shown to cause a reduction in chloroplast number in
carrot callus cultures (Edelman and Hanson 1971). However, similar studies on intact, developing higher plants,
such as Arabidopsis thaliana, have been lacking.
To test whether chloroplast development is inhibited
in seedlings grown on Glc, the rate of accumulation of a
chloroplast-specific fatty acid was measured in seedlings
grown on a variety of media. A chloroplast-specific fatty
acid (16:3), rather than a chloroplast-specific protein,
was chosen to monitor chloroplast development as fatty
acid assays are not only sensitive, but, in comparison to
such typical methods of protein quantification as western
blots and immunofluorescence, require relatively little
labour and are highly quantitative and reproducible. In
addition, 16:3 comprises a major structural component
of chloroplast membranes (Harwood 1980). Consequently,
16:3 levels are expected to reflect the total amount of
chloroplast membranes and therefore the total amount
of chloroplast material.
The results presented herein indicate that chloroplast
development is impaired in Arabidopsis thaliana seedlings
grown in the presence of exogenous Glc. Seedlings grown
on moderate (e.g. 0.14 M) concentrations of Glc exhibit a
significant decrease in the rate of 16:3 accumulation
relative to seedlings grown on low (e.g. 0.03 M) or no
Glc. The effects of 0.14 M Glc on 16:3 levels are not
solely due to alterations in the osmotic potential of the
media, as equi-molar and even twice equi-molar concentrations of sorbitol have less effect on 16:3 levels. The
effects of high (e.g. 0.3 M) Glc on 16:3 accumulation
were also determined. These experiments indicate that
seedlings grown on high-Glc media accumulate almost
no 16:3, even after 22 days of growth under continuous
light conditions. To determine whether the very low 16:3
levels found in Glc-arrested seedlings do reflect a lack of
462
chloroplasts, transmission electron microscopy was used
to examine cells from plants grown on high Glc. These
experiments indicate that cells from the cotyledons of
Glc-arrested seedlings contain no structures resembling
mature chloroplasts.
Seedlings arrested by growth on high Glc accumulate
lower levels of all major membrane fatty acid species
than seedlings grown on low or no Glc. However,
Glc-arrested seedlings exhibit a greater percentage reduction in net accumulation of the chloroplast-specific fatty
acid 16:3 than in accumulation of fatty acids, such as
16:0, 18:2 and 18:3, that are found in both chloroplast
and non-chloroplast membranes. Therefore, the effect of
exogenous Glc on 16:3 levels does exhibit a degree of
specificity and is not simply the result of an overall
reduction in seedling growth rates.
The mechanism by which chloroplast development is
impaired in the presence of exogenous Glc remains to be
determined. Interestingly, Arabidopsis seedlings become
insensitive to the inhibitory effects of exogenous Glc on
chloroplast formation within approximately 3 days of
the start of imbibition. In contrast, Arabidopsis seedlings
remain sensitive to the inhibitory effects of 0.4 M sorbitol
1 0.03 M Glc, providing further evidence that the effects
of high concentrations of Glc on chloroplast development can not be due solely to the osmotic potential of the
media. The finding that seedlings become insensitive to
the effects of Glc on chloroplast development within
3 days of the start of imbibition also suggests that
completion of some critical developmental or metabolic
transition results in loss of sensitivity to Glc-mediated
inhibition of chloroplast development. High concentrations of exogenous Glc might inhibit chloroplast development by repressing expression of gene(s) that play
critical roles in the formation of mature chloroplasts
from the proplastids found in plant embryos (Bauer et al.
2001). Numerous plant genes are known to be regulated
by sugar levels (Koch 1996). Alternatively, the effects of
Glc on chloroplast development could be relatively indirect.
In any case, the work presented here indicates that
sugar levels can strongly affect organelle biogenesis
not only in unicellular organisms and callus cultures,
but also in intact higher plants.
Acknowledgements – We thank Dr David Caprette for providing
helpful instruction in the preparation and visualization of transmission
electron microscopy samples. This work was supported by the US
Department of Energy, Energy Biosciences Program Grants
DE-FG03–00ER15061 (S.I.G) and DE-FG02–95ER20203 (W.-D.R).
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Edited by W. S. Chow
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