Download Loss of Starch Granule Initiation Has a

Loss of Starch Granule Initiation Has a Deleterious Effect
on the Growth of Arabidopsis Plants Due to an
Accumulation of ADP-Glucose1[W]
Paula Ragel, Sebastian Streb, Regina Feil, Mariam Sahrawy, Maria Grazia Annunziata, John E. Lunn,
Samuel Zeeman, and Ángel Mérida*
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas-University
of Sevilla, 41092 Seville, Spain (P.R., A.M.); Department of Biology, Eidgenössische Technische Hochschule
Zürich, CH–8092 Zurich, Switzerland (S.S., S.Z.); Estación Experimental del Zaidín, Consejo Superior de
Investigaciones Científicas, 18008 Granada, Spain (M.S.); and Max Planck Institute of Molecular Plant
Physiology, D–14476 Potsdam-Golm, Germany (R.F., M.G.A., J.E.L.)
STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3
can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under longday conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There
is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters
and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose
(Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1)
or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4
and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the
plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic
and stunted growth phenotypes of the plants.
The metabolism of starch plays an essential role in
the physiology of plants. Starch breakdown provides
the plant with carbon skeletons and energy when the
photosynthetic machinery is inactive (transitory starch)
or in the processes of germination and sprouting (storage starch). Deficiencies in the accumulation of transitory starch in Arabidopsis (Arabidopsis thaliana) have
been described previously, specifically in mutants affected in the plastidial phosphoglucomutase (PGM1) or
the small subunit (APS1) of the ADP-Glc pyrophosphorylase (AGPase). While they are described as
“starchless,” they actually contain small amounts of
starch (1%–2% of the wild-type levels; Streb et al.,
2009) and share similar phenotypic alterations, such as
growth retardation when cultivated under a short-day
photoregime and increased levels of soluble sugars
1
This work was supported by the Comisión Interministerial de
Ciencia y Tecnología and the European Union-FEDER (grant no.
BIO2009–07040), by the Junta de Andalucía (grant nos. P07–CVI–
02795 and P09–CVI–4704), and by the Spanish Ministry of Education
(Formación de Personal Universitario grant to P.R.).
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Ángel Mérida ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.113.223420
during the light phase and reduced levels during the
night (Caspar et al., 1985; Lin et al., 1988b; Schulze
et al., 1991). Carbon partitioning is altered in these
plants. As photosynthate cannot be accumulated as
starch, it is diverted via hexose phosphates in the cytosol to the synthesis of Suc, which accumulates together
with the hexose sugars, Glc and Fru (Caspar et al., 1985).
In Arabidopsis, there are five starch synthase isoforms:
one granule-bound starch synthase and four soluble
starch synthases: SS1, SS2, SS3, and SS4. We have described previously an Arabidopsis mutant plant lacking SS3 and SS4 that is also severely affected in the
accumulation of starch (Szydlowski et al., 2009). SS4 is
involved in the initiation of the starch granule and
controls the number of granules per chloroplast (Roldán
et al., 2007). The elimination of SS3 in an ss4 background leads to an absence of starch in most of the
chloroplasts, despite the fact that SS1 and SS2 are still
present and total starch synthase activity is only reduced by 35% (Szydlowski et al., 2009). However, a
very small proportion of chloroplasts of this mutant
plant contain a single huge starch granule, which is also
a characteristic of chloroplasts in the ss4 single mutant
(D’Hulst and Mérida, 2012). Thus, like aps1 and pgm1,
ss3/ss4 plants contain only small amounts of starch.
However, unlike aps1 or pgm1 plants, most of the cells
of this mutant have empty chloroplasts, without starch
(Szydlowski et al., 2009).
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Ragel et al.
In this work, we have analyzed the phenotypic effects of the impaired starch accumulation of ss3/ss4
plants. We show that this mutant displays phenotypic changes that are not found in other mutants
with very low levels of starch, such as aps1 or pgm1
plants. We provide evidence that extremely high
levels of ADP-Glc accumulate in the ss3/ss4 plants.
Using reverse genetics to block the pathway of starch
synthesis upstream of the starch synthases reduced
the level of ADP-Glc in ss3/ss4 plants and reverted
the other phenotypic traits. This suggests that ADPGlc accumulation is the causal factor behind the chlorotic and stunted growth phenotypes of the ss3/ss4
mutant.
RESULTS
Phenotypic Characterization of an ss3/ss4 Double Mutant
Arabidopsis plants mutated in both SS4 and SS3
genes accumulate very low levels of starch in leaves
(around 12% of wild-type values; Fig. 1A) but have
slightly higher levels of soluble sugars, particularly
Suc (Fig. 1, B–D). However, this double mutant plant
displays some phenotypic effects, such as poor growth
rate under a long-day photoregime and pale green
color (Fig. 2), that are not observed in other starch
synthase-deficient mutants (Delvallé et al., 2005; Zhang
et al., 2005) or in mutants that accumulate very low
amounts of starch, such as those affected in APS1 (Lin
Figure 1. Starch, sugar, and adenine
nucleotide contents of wild-type and
mutant Arabidopsis rosettes. Wild type
(Col-0), ss3, ss4, ss3/ss4, aps1, pgm1,
aps1/ss3/ss4, and pgm1/ss3/ss4 plants
were grown under LD conditions (16 h
of light, 20˚C/8 h of dark, 18˚C) and an
irradiance of 150 mE m22 s21. Plants
were harvested at midday at the nineleaf stage and processed as described
in “Materials and Methods.” Eight to
10 plants per sample were used for the
ss3/ss4 mutant and four to five plants
for the other genotypes. Values represent means 6 SD of determinations on
six independent samples (three independent samples for measurements of
starch, sugars, and ADP-Glc in ss3s/
ss4). Letters indicate samples that were
not significantly different (P $ 0.05)
according to one-way ANOVA (HolmSidak test). There were no significant
differences in AMP content between
the eight genotypes. ADPG, ADP-Glc;
FW, fresh weight.
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Growth Defects of ss3/ss4 Linked to High ADP-Glucose
starch and showed essentially the same growth rate as
aps1 (data not shown). Figure 2 shows that aps1, ss4,
and ss3/ss4 plants all grew more slowly than wild-type
plants in the SD condition, with growth being most
severely restricted in the ss3/ss4 mutant. In contrast,
the ss3 mutant grew slightly faster than wild-type
plants. Increasing the length of the light period greatly
increased the growth of the aps1 mutant, which was
restored to nearly wild-type levels in LL conditions
(Fig. 2). However, the growth of ss3/ss4 in LD and LL
conditions remained severely limited, while the phenotype of ss4 was intermediate between that of ss3/ss4
and wild-type plants (Figs. 2 and 3). These results indicate that the poor growth of ss3/ss4, and to a lesser
extent of ss4, is not caused solely by the impairment in
starch accumulation and that some other metabolic
perturbation, which is specific to these mutants, restricts their growth.
Photosynthetic Activity
The low growth rate observed in ss4 and ss3/ss4
plants suggests that their photosynthetic activity is
negatively affected by the defects in starch synthesis.
To investigate which aspects of photosynthesis are affected, CO2 assimilation rate versus photosynthetically
active radiation were determined on attached leaves
of plants grown under a LD photoregime at 130 mmol
m22 s21 and ambient CO2 using an open gas-exchange
system. The light-response curves are shown in Figure 4.
The light-saturated photosynthesis rates of ss4 (6.87 6
0.39 mmol CO2 m22 s21) and ss3/ss4 (2.51 6 0.52 mmol
Figure 2. Effects of photoperiod on growth phenotypes of starch-deficient
mutants. Plants were grown in growth cabinets under different photoperiods: LD (16 h of light/8 h of dark), SD (8 h of light/16 h of dark), and
LL. The growth of the different mutants was documented by photographs
of representative 3-week-old plants. WT, Wild type.
et al., 1988a; Fig. 1A) or in the chloroplastic PGM1
(Caspar et al., 1985; Fig. 1A). We have carried out a
comprehensive phenotypic analysis of the ss3/ss4 mutant
in order to identify the metabolic cause of its distinctive
growth and morphological traits.
Growth of the ss3/ss4 Mutant
Plants were cultivated in controlled conditions under
different photoregimes: long day (LD; 16 h of light/8 h
of dark), short day (SD; 8 h of light/16 h of dark), and
continuous light (LL), and their growth was documented by photographs of 21-d-old plants (Fig. 2) and
by a time course of the growth of plants cultivated
under LD conditions (Fig. 3). The Arabidopsis aps1
mutant, which lacks the small subunit of AGPase and
accumulates only very low amounts of starch, was used
as a control, and its growth rate was compared with
those of ss3, ss4, ss3/ss4, and wild-type plants. The
pgm1 mutant also accumulated very low amounts of
Figure 3. Growth rates of wild-type (WT) Col-0, ss3, ss4, and ss3/ss4
plants. Plants were grown in growth cabinets under a LD photoperiod
(16 h of light/8 h of dark). Aerial parts of the plants (five plants per
sample) were weighed on the days indicated. Each point represents the
mean 6 SD of three independent experiments.
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Ragel et al.
antenna and accessory pigments (chlorophylls a and b
and carotenoids) were about 45% lower in the double
mutant than in wild-type plants. There was a smaller
decrease, to around 63% of the wild type, in the ss4
single mutant. While chlorophyll and carotenoid levels
were decreased, the ss4 and ss3/ss4 mutants accumulated more anthocyanins than the controls, with 1.4- and
5.8-fold higher levels than wild-type plants, respectively
(Fig. 5). The pigment content of the ss3 mutant was not
significantly different from the wild type.
Photooxidative Stress
Figure 4. Photosynthetic assimilation rates. Light-response curves of
CO2 assimilation rates (A) of wild-type (WT), ss3, ss4, and ss3/ss4
plants were measured in a leaf chamber. Three-week-old plants grown
in growth cabinets under a LD photoperiod were used to determine the
CO2 assimilation rates. Each point represents the mean 6 SD of five
independent determinations.
CO2 m22 s21) in these conditions were substantially
lower (65% and 24% of the control rate, respectively)
than the maximum value obtained for wild-type plants
(10.57 6 0.64 mmol CO2 m22 s21). Values obtained for
the ss3 mutant plants (10.63 6 0.32 mmol CO2 m22 s21)
were not significantly different from wild-type plants
(Fig. 4). Measurements of modulated chlorophyll a
fluorescence (see “Materials and Methods”) indicated
that the maximum quantum efficiency of PSII photochemistry (Fv/Fm) was drastically reduced in the ss3/ss4
mutant (0.63 6 0.040 and 0.82 6 0.002 for ss3/ss4 and
wild-type plants, respectively; Table I). A reduction was
also observed in the PSII operating efficiency (FPSII),
indicating a decrease in the relative quantum yield of
linear electron transfer through the photosystems (Baker,
2008). The values of the parameters obtained for ss4
mutant plants were also significantly different from those
obtained for wild-type plants (Table I), being intermediate between wild-type and ss3/ss4 double mutant plants.
This suggests that the single ss4 mutation leads to negative effects on both PSII efficiency and the CO2 assimilation rate of the plant, which are exacerbated by loss of
the SS3 gene function.
Reducing the levels of antenna pigments would be
expected to help balance the input and utilization of
light energy in the ss4 and ss3/ss4 mutants. Nevertheless, despite this adaptive response, blockage of the
electron flux between photosystems could result in
photooxidative damage in these plants. Photooxidative
damage caused by reactive oxygen species (ROS) in ss4
and ss3/ss4 was confirmed by their higher content of
malondialdehyde (MAD; Fig. 6), which is a marker of
membrane lipid peroxidation caused by elevated levels
of ROS (Janero, 1990). The MAD content was 2.5 times
higher in ss3/ss4 than in wild-type plants, while ss4
plants had levels intermediate between ss3/ss4 and the
wild type, being 1.5 times higher than wild-type plants
(Fig. 6).
The ss3/ss4 and ss4 Mutants Accumulate ADP-Glc
ADP-Glc is the substrate for starch synthesis. Therefore, impairment of starch synthesis as found in ss4 and
ss3/ss4 mutants (Roldán et al., 2007; Szydlowski et al.,
2009) might be expected to affect the accumulation of
ADP-Glc, especially in the ss3/ss4 mutant, which has
very low levels of starch. ADP-Glc was measured in
wild-type and mutant plants by high-performance anionexchange chromatography coupled to tandem mass
spectrometry (MS/MS; Lunn et al., 2006). This method
provides greater specificity and sensitivity than the
HPLC-based methods with UV detection that have
Table I. Chlorophyll a fluorescence parameters of wild-type, ss3, ss4,
ss3/ss4, aps1, and aps1/ss3/ss4 plants
Pigment Content
The lower yield of linear electron flux through the
photosystems observed in ss3/ss4 and ss4 mutant plants
could trigger some adaptive responses, such as a decrease in the light-harvesting antenna size, in order to
prevent possible photooxidative damage (Niyogi, 1999).
Such a response would account for the pale green color
that is characteristic of ss4 and ss3/ss4 mutants compared with the other starch-deficient mutants. To test
this hypothesis, we measured the levels of different
pigments in ss3/ss4 plants and compared them with the
pigment contents of both the single parental mutants
and wild-type plants. Figure 5 shows that the levels of
Fv /Fm and FPSII at 130 mE m22 s21 were determined in dark-adapted
plants. Values are means 6 SE of four independent determinations.
Significance is indicated as follows: a, not significantly different from
the wild type according to Student’s t test (P $ 0.01); b, significantly
different from the wild type according to Student’s t test (P , 0.01).
Genotype
Fv /Fm
FPSII
Wild type (Col-0)
ss3
ss4
ss3/ss4
aps1
aps1/ss3/ss4
0.816 6 0.001
0.801 6 0.001 a
0.754 6 0.008 b
0.631 6 0.040 b
0.803 6 0.003 a
0.802 6 0.003 a
0.513 6 0.000
0.483 6 0.008 a
0.350 6 0.006 b
0.220 6 0.044 b
0.480 6 0.010 a
0.478 6 0.027 a
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Growth Defects of ss3/ss4 Linked to High ADP-Glucose
the plastidial enzymes that synthesize ADP-Glc and
Glc-1-P, respectively. The levels of Suc, Fru, and Glc in
ss3/ss4 plants were lower than in aps1 or pgm1, which
contained 2- to 3-fold higher soluble sugar levels than
wild-type plants (Fig. 1, B–D). Measurements of other
metabolites by high-performance anion-exchange chromatography coupled to MS/MS showed a 2-fold higher
level of trehalose 6-phosphate in ss3/ss4 compared with
the wild type (Supplemental Fig. S1), presumably reflecting the 2-fold higher Suc content of the mutant (Fig. 1D).
There were no striking differences between ss3/ss4 and
the wild type in the levels of other sugar phosphates or
glycolytic intermediates, apart from a decrease in
Fru-1,6-bisP in the mutant (Supplemental Fig. S1).
Citrate, aconitate, and isocitrate were slightly elevated
in the mutant, while the other tricarboxylic acid cycle
intermediates were either unchanged (2-oxoglutarate
and succinate) or slightly lower (malate and fumarate)
in the ss3/ss4 mutant (Supplemental Fig. S1).
Restricting Plastidial ADP-Glc Synthesis Substantially
Restores Growth in the ss3/ss4 Plants
The high levels of ADP-Glc found in ss3/ss4 plants
are presumably caused by an imbalance between the
rates of ADP-Glc synthesis by AGPase and the consumption for starch synthesis, which is essentially
blocked in this mutant. As a result, it seems likely
that much of the adenine nucleotides in the chloroplasts will be effectively locked away in this large pool
of ADP-Glc, restricting photophosphorylation, CO2
Figure 5. Photosynthetic pigments and anthocyanin contents. Plants
were grown in growth cabinets under a LD photoperiod, and discs of
rosette leaves from four different plants per sample were used to determine the chlorophyll a and b, carotenoid, and anthocyanin contents
of wild-type (WT), ss3, ss4, and ss3/ss4 plants (A) and wild-type, aps1,
pgm1, ss3/ss4, aps1/ss3/ss4, and pgm1/ss3/ss4 plants (B). Values are
means 6 SD of three independent experiments. Asterisks indicate
values that are significantly different from the wild type according to
Student’s t test (P , 0.05). fw, Fresh weight.
generally been used to measure ADP-Glc in plants.
The ss3/ss4 plants were found to have extremely high
levels of ADP-Glc (341 nmol g21 fresh weight), which
was around 170-fold higher than in wild-type plants
(Fig. 1E). The content of this compound in other mutants that accumulate only trace amounts of starch,
such as aps1 and pgm1, was lower than in wild-type
plants (Fig. 1E) as a consequence of the mutations in
Figure 6. MAD accumulation. MAD content in leaves of wild-type
(WT), ss3, ss4, and ss3/ss4 plants grown under a LD photoperiod was
determined using the thiobarbituric acid method. Values are means 6
SD of four independent experiments. Asterisks indicate values that are
significantly different from the wild type according to Student’s t test
(P , 0.05).
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Ragel et al.
fixation, and other process that are dependent on ATP.
To test this hypothesis, we crossed ss3/ss4 plants with
mutant plants lacking the small subunit of AGPase
(aps1) or the plastidial isoform of PGM (pgm1). Both
mutants are greatly impaired in the synthesis of plastidial ADP-Glc and accumulate only very small amounts
of starch (Stitt and Zeeman, 2012; Fig. 1A). Progeny of the
respective crosses were analyzed to identify aps1/ss3/ss4
and pgm1/ss3/ss4 triple mutants (see “Materials and
Methods”), and these plants were selected for subsequent analyses.
Mutant plants cultured under a 16-h-light/8-h-dark
(aps1/ss3/ss4) or a 12-h-light/12-h-dark (pgm1/ss3/ss4)
photoregime showed the same growth rate as the aps1
or pgm1 parental plants, respectively, which were higher
than the growth rate of ss3/ss4 parental plants (Fig. 7;
Supplemental Fig. S2). Attached leaves of aps1/ss3/ss4
plants or whole pgm1/ss3/ss4 plants grown under the
same conditions detailed above were used to determine
their CO2 assimilation rates. Under saturating light, the
CO2 assimilation rates of the ss3/ss4 (2.51 6 0.52 mmol
CO2 m22 s21) and aps1 (6.08 6 0.23 mmol CO2 m22 s21)
mutants were 61% and 36% lower, respectively, than in
wild-type plants (9.29 6 1.01 mmol CO2 m22 s21; Fig. 8).
The CO2 assimilation rate of aps1/ss3/ss4 plants (6.69 6
0.77 mmol CO2 m22 s21) was similar to that found for
aps1 plants, indicating that the nearly complete loss of
AGPase activity overrides the severe inhibition of photosynthesis in the ss3/ss4 mutant background (Fig. 8).
Similarly, restricting ADP-Glc synthesis via the loss of
plastidial PGM activity restored photosynthetic rates
in the pgm1/ss3/ss4 mutant to wild-type levels (Fig. 9).
Measurements of Fv/Fm and FPSII showed that both
of these photosynthetic parameters in the aps1/ss3/ss4
and pgm1/ss3/ss4 triple mutants had reverted to wildtype values (Tables I and II). The levels of photosynthetic pigments (chlorophylls a and b and carotenoids)
in aps1/ss3/ss4 were similar to those of wild-type plants
(Fig. 5), providing further evidence that restricting
ADP-Glc counteracted the negative effects of the loss
of SS3 and SS4 activity on photosynthesis. Levels of
anthocyanins in the aps1/ss3/ss4 mutant plants were
much lower than in the parental ss3/ss4 mutant, being
slightly higher than in wild-type plants but similar to
the parental aps1 mutant (Fig. 5). Metabolite profiling
of the triple mutants revealed that they accumulated
87% to 97% less ADP-Glc than the ss3/ss4 mutant (Fig. 1E).
However, the levels of ADP-Glc in aps1/ss3/ss4 plants
were still considerably higher (around 23-fold) than in
wild-type plants but only about 5-fold higher than the
wild type in the pgm1/ss3/ss4 mutant.
To assess the impact of the massive accumulation of
ADP-Glc on other adenine nucleotides, we measured
ATP, ADP, and AMP in mutant and wild-type plants.
ATP was about 27% lower in the ss3/ss4 mutant than in
wild-type plants (Fig. 1F). The difference was statistically significant (P = 0.047) according to Student’s t test
but not significant according to one-way ANOVA
(Holm-Sidak test; Fig. 1F). ADP also showed a tendency
to be decreased in the ss3/ss4 mutant (Fig. 1G). Both
Figure 7. Growth phenotypes of wild-type (WT), aps1, pgm1, ss3/ss4,
aps1/ss3/ss4, and pgm1/ss3/ss4 plants. The photographs show 3-weekold plants cultivated in growth cabinets under a 16-h-light/8-h-dark
(aps1, ss3/ss4, and aps1/ss3/ss4) or a 12-h-light/12-h-dark (pgm1, ss3/ss4,
and pgm1/ss3/ss4) photoperiod.
ATP and ADP were significantly lower in the pgm
mutant than in the wild type (Fig. 1F). The total adenine nucleotide pools (ATP + ADP + AMP + ADP-Glc)
in the ss3/ss4 and ss4 mutants were about four times
higher than in wild-type plants, with ADP-Glc constituting 81%, 77%, and 2% of the total adenylate pool,
respectively (Fig. 1; Supplemental Fig. S3). The ATP,
ADP, and total adenylate pool sizes in the aps1/ss3/ss4
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Growth Defects of ss3/ss4 Linked to High ADP-Glucose
Figure 8. Photosynthetic assimilation rates. Light-response curves
of CO2 assimilation rates (A) of wild-type (WT), aps1, ss3/ss4, and
aps1/ss3/ss4 plants were measured in a leaf chamber. Three-week-old
plants grown in growth cabinets under a LD photoperiod were used
to determine the CO2 assimilation rates. Each point represents the
mean 6 SD of five independent determinations.
and pgm/ss3/ss4 triple mutants were indistinguishable
from the wild-type levels (Fig. 1, F and G; Supplemental
Fig. S3).
DISCUSSION
We have previously reported that Arabidopsis requires SS4 or SS3 to synthesize starch (Szydlowski
et al., 2009), strongly implicating SS4 and SS3 in the
process of granule initiation. In subsequent analyses of
the ss3/ss4 mutant, we have observed that a small
number of chloroplasts do contain single starch granules with a distinctive shape, being more rounded than
the discoid granules usually found in Arabidopsis
leaves (D’Hulst and Mérida, 2012). This suggests that
in the absence of SS4, stochastic initiation of starch
granules can still occur, albeit very infrequently, even
when SS3 is also missing (D’Hulst and Mérida, 2012).
This would explain the trace amounts of starch in the
ss3/ss4 plants (Fig. 1A). The most obvious phenotypic
traits of the ss3/ss4 plants are their severely restricted
growth and chlorotic leaves (Figs. 2 and 3). Transitory
starch reserves provide the plant with a buffer itself
against diurnal variation in carbon and energy supplies
(Sun et al., 1999; Smith and Stitt, 2007). Mutants impaired in starch synthesis, such as aps1 and pgm1, accumulate high levels of Suc and hexoses (Caspar et al.,
1985) as a consequence of their inability to store net
photosynthate in starch. However, the total amount of
carbohydrate accumulated as soluble sugars in starchdeficient mutants during the day is much less than the
amount of starch accumulated by wild-type plants
(Fig. 1, A–D). Furthermore, soluble sugars are directly
available for export and respiration, so these reserves,
which are already smaller than those in wild-type
plants, are rapidly depleted during the first hours of
the night. This leads to carbon starvation by the end of
the night, which suppresses growth by mechanisms
that are not yet fully understood but probably involve
the inhibition of translation and other biosynthetic processes and wasteful turnover of proteins (Gibon et al.,
2004). The carbon starvation diminishes as the length
of the night period is reduced; hence, the growth rates
of the aps1 (Fig. 2) and pgm1 mutant plants in LD or LL
conditions were practically the same as that observed
in wild-type control plants (Lin et al., 1988a). The ss3/ss4
plants show a severe dwarf phenotype even in LD or
LL photoregimes (Figs. 2 and 3), suggesting that the
impairment in starch synthesis on its own is not the
underlying cause of the poor growth of these plants and
that other metabolic changes must be responsible for
their stunted phenotype.
In chloroplasts, the substrate for starch synthesis is
derived from the Calvin-Benson cycle in the form of
Fru-6-P. This is converted to Glc-6-P and then Glc-1-P
in two reversible reactions catalyzed by phosphoglucose
isomerase and phosphoglucomutase, respectively. The
next step in the pathway, the synthesis of ADP-Glc by
AGPase, is rendered irreversible by the hydrolysis of
the coproduct, pyrophosphate, by inorganic pyrophosphatase (Ballicora et al., 2003). Thus, loss of plastidial
phosphoglucose isomerase, PGM, or AGPase activity
essentially blocks both ADP-Glc and starch synthesis,
although a small amount of starch may be synthesized
from imported hexose phosphate or ADP-Glc in young
developing leaves, bypassing the metabolic lesions in
Figure 9. Photosynthetic assimilation rates. Whole, 3-week-old plants
were used to determine the photosynthetic CO2 assimilation rates (A)
in a multichamber system at an irradiance of 150 mE m22 s21. Values
are means 6 SD of four biological replicates. WT, Wild type.
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Ragel et al.
Table II. Chlorophyll a fluorescence parameters of wild-type, ss3, ss4,
ss3/ss4, pgm1, and pgm1/ss3/ss4 plants
Fv /Fm and FPSII at 130 mE m22 s21 were determined in dark-adapted
plants. Values are means 6 SE of four independent determinations.
Significance is indicated as follows: a, not significantly different from
the wild type according to Student’s t test (P $ 0.01); b, significantly
different from the wild type according to Student’s t test (P , 0.01).
Genotype
Fv /Fm
FPSII
Wild type (Col-0)
ss3
ss4
ss3/ss4
pgm1
pgm1/ss3/ss4
0.808 6 0.0005
0.803 6 0.0015 a
0.771 6 0.0015 b
0.726 6 0.005 b
0.790 6 0.000 a
0.779 6 0.0005 a
0.394 6 0.009
0.414 6 0.003 a
0.315 6 0.002 b
0.204 6 0.005 b
0.381 6 0.003 a
0.391 6 0.006 a
these mutants (Streb et al., 2009; Bahaji et al., 2011).
The situation is different in the ss3/ss4 mutant, where
starch synthesis is limited not by the availability of
ADP-Glc but by the almost complete failure of starch
granule initiation and the consequent lack of primers
for glucan synthesis. Given the pyrophosphatasedriven irreversibility of the AGPase reaction, there is
no obvious reason why ADP-Glc should not continue
to be synthesized. Starch synthesis is the main route
for catabolizing ADP-Glc in the chloroplasts (Ball et al.,
2011), but as this route is blocked in the ss3/ss4 mutant,
there is likely to be an imbalance between ADP-Glc
production and consumption, leading to its accumulation in the chloroplast stroma. Metabolite profiling
confirmed that the ss3/ss4 mutant does indeed accumulate massive amounts of ADP-Glc (341 6 16 nmol g21
fresh weight), reaching levels that are over 170 times
higher than in wild-type plants (Fig. 1E). In addition,
levels of soluble sugars are only slightly increased in
the ss3/ss4 mutant (Fig. 1, B–D), likely caused by the
severe decrease of CO2 fixation in this mutant, which
determines that the carbon diverted to soluble sugars,
such as Suc, as consequence of the blockage of starch
synthesis is lower than in aps1 or pgm1 mutants.
The unprecedented accumulation of ADP-Glc in the
ss3/ss4 plants will have important knock-on effects in
the chloroplasts, as the adenosine and phosphate moieties within the ADP-Glc pool will in effect be locked
away and unavailable for other metabolic processes. In
wild-type plants, ATP and ADP are the dominant adenine nucleotides, with AMP and ADP-Glc forming
only a small percentage (about 2% each) of the total
adenine nucleotide pool (Fig. 1, E–H; Supplemental
Fig. S3). Our measurements of ATP and ADP in wildtype plants indicate a total pool size of around 100
nmol g21 fresh weight, close to the value of 120 nmol g21
fresh weight reported by Strand et al. (2000). This is
actually lower than the amount of ADP-Glc alone that
we found in the ss3/ss4 and ss4 mutants, and the total
adenylate pools in these two mutants are four times
higher than in wild-type plants (Supplemental Fig. S3).
This suggests that there has been a massive compensatory increase in the total adenine nucleotide pool size
in the mutants, but despite this increase, the vast majority
(77%–81%) of the enlarged pools are still sequestered
in the form of ADP-Glc. As a consequence, there will
be very little ADP available in the chloroplast stroma
for photophosphorylation, which in turn will have
severely negative effects on many metabolic and other
processes in the chloroplasts.
The most obvious effect of impaired ATP synthesis
will be the limitation of photosynthetic CO2 assimilation, as observed in the ss3/ss4 mutant (Fig. 4). Within
the Calvin-Benson cycle, ATP is required not only for
the regeneration of ribulose-1,5-bisphosphate but also for
the synthesis of triose phosphates from 3-phosphoglycerate
(Heldt and Heldt, 2006; MacRae and Lunn, 2006). Limiting
the synthesis of triose phosphates will, in turn, restrict
their export to the cytosol for the synthesis and export
of Suc to sink organs such as developing leaves, roots,
flowers, and seeds. Low stromal ATP levels will also
inhibit many of the biosynthetic pathways that occur
in the chloroplasts (e.g. for the production of lipids,
amino acids, nucleotides, isoprenoids, phenylpropanoids,
and vitamins; Lunn, 2007). Presumably, at some point,
ATP will become limiting for the further synthesis of
ADP-Glc itself. However, the Km (ATP) of the Arabidopsis AGPase is around 70 mM (Crevillén et al., 2003),
so it is capable of drawing down the stromal concentration of ATP to very low levels. ATP is also required
for many housekeeping functions in the chloroplasts,
including DNA, RNA, and protein synthesis, as well as
the import, processing, and assembly of nucleus-encoded
proteins. Altogether, the profound disturbance of chloroplast metabolism caused by low ATP availability is
bound to have far-reaching effects beyond the chloroplasts, reducing the energy and carbon supplies available for cell maintenance and growth.
Low ADP availability in the chloroplasts could also
result in damage to existing chloroplast structures due
to overenergization of the thylakoid membranes. The
reduced rate of photophosphorylation and, therefore,
dissipation of the proton-motive force across the thylakoid membranes will lead to hyperpolarization of
the membranes, blocking further electron transport
between the photosystems (Heldt and Heldt, 2006).
This will lower the relative quantum yield of the linear
electron transport, leading to a reduction in FPSII, as
observed in the ss3/ss4 plants (Table I; Baker, 2008).
This situation favors the formation of ROS that can
cause oxidative damage to the photosynthetic apparatus (Niyogi, 1999). This is corroborated by the low
value of Fv/Fm found in the ss3/ss4 plants, which is
indicative of photoinhibition (Maxwell and Johnson,
2000). Further evidence of oxidative stress in the ss3/ss4
plants came from the finding of high levels of MAD in
these plants, which is a characteristic product of membrane lipid peroxidation by ROS (Janero, 1990). One of
the adaptive responses of plants to photoinhibitory
damage is the adjustment of the light-harvesting antenna
size in order to balance light absorption and utilization
(Niyogi, 1999). This response explains the reduction in
the amounts of chlorophylls a and b and carotenoids
found in the ss3/ss4 plants (Fig. 2). Finally, the high levels
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Growth Defects of ss3/ss4 Linked to High ADP-Glucose
of anthocyanins detected in the ss3/ss4 plants provide
further support for the conclusion that the ss3/ss4 plants
are subject to photoinhibition. Anthocyanin accumulation generally coincides with situations where there is
an imbalance between light capture, CO2 assimilation,
and carbohydrate utilization (Wand et al., 2002).
All of the phenotypic traits described for the ss3/ss4
plants were also detected in the parental ss4 single
mutant, such as pale color (Fig. 2; Roldán et al., 2007),
lower growth and CO2 assimilation rates (Figs. 3 and 4),
and low values of Fv/Fm and FPSII (Table I), although
these effects were less severe than in the ss3/ss4 plants.
Plants lacking SS4 accumulate around 60% to 80% of
the starch found in wild-type plants (Roldán et al.,
2007), suggesting that there is greater turnover of the
ADP-Glc pool in ss4 than in the ss3/ss4 double mutant.
Nevertheless, we observed a similarly large accumulation of ADP-Glc in both mutants, indicating that the
limitation of starch synthesis in the single ss4 mutant is
sufficient to have a major impact on ADP-Glc levels and
to trigger a similar compensatory increase in the total
adenylate pool to that seen in the ss3/ss4 mutant.
We tested the hypothesis that the chlorotic and growth
phenotypes of the ss3/ss4 mutant are attributable to the
massive ADP-Glc accumulation by introducing further
mutations (aps1 and pgm1) to limit ADP-Glc synthesis.
The pgm1 mutation blocks the production of Glc-1-P in
the chloroplasts, so there is no substrate available for
ADP-Glc synthesis, except for a very small amount
imported from the cytosol by a Glc-1-P transporter
(Fettke et al., 2011). Introduction of the pgm1 mutation
into the ss3/ss4 background lowered the amount of
ADP-Glc accumulated in the leaves by 97% and restored
the photosynthetic capacity and growth of the plants to
nearly wild-type levels (Table II; Figs. 1E, 5, and 7–9).
Knocking out the APS1 gene, encoding the small subunit
of AGPase, led to an 87% decrease in the accumulation of
ADP-Glc in the ss3/ss4 background but was less effective
than the loss of PGM activity (Fig. 1E). Nevertheless, the
aps1/ss3/ss4 triple mutant grew much better than the ss3/ss4
double mutant and was phenotypically similar to the aps1
parent (Table I; Figs. 5, 7, and 8).
Loss of the APS1 subunit does not appear to abolish
AGPase activity entirely, because the aps1 mutant was
found to contain low but detectable traces of ADP-Glc
and a small amount of starch (about 2% of the wildtype level; Fig. 1A). The residual activity can be ascribed
to the remaining AGPase large subunits (APL1–APL4),
several of which have been demonstrated to have a low
level of catalytic activity (Ventriglia et al., 2008). Metabolites were measured in plants harvested after 8 h of
illumination. If we assume that all of the ADP-Glc in the
aps1/ss3/ss4 mutant (44 nmol g21 fresh weight; Table I)
had been synthesized during this period and that there
was negligible consumption of ADP-Glc for starch
synthesis, we can calculate that as little as 0.09 nmol
min21 g21 fresh weight of AGPase activity would be
sufficient to account for the observed accumulation of
ADP-Glc in the triple mutant. This level of activity
would represent less than 0.01% of the maximal catalytic
activity of AGPase in wild-type Arabidopsis rosettes
(1.0–1.5 mmol min21 g21 fresh weight; Hädrich et al.,
2012). The amount of ADP-Glc in the aps1/ss3/ss4
mutant constitutes 34% of the total adenylate pool (Fig.
1E; Supplemental Fig. S3). Thus, by limiting the accumulation of ADP-Glc to a moderate level, a substantial
pool of ATP and ADP should remain available for
photophosphorylation and other biochemical reactions
in the aps1/ss3/ss4 mutant. The photosynthetic capacities and growth rates of the aps1/ss3/ss4 and aps1
mutants were very similar, indicating that even such a
partial rebalancing of the adenine nucleotide pool was
sufficient to support higher rates of CO2 fixation and
growth. Loss of plastidial PGM activity was even more
effective at restoring the adenine nucleotide and ADPGlc pools of the pgm/ss3/ss4 mutant to nearly wild-type
levels (Fig. 1, E–H).
In conclusion, the loss of SS3 and SS4 activity in
Arabidopsis leaves almost totally abolishes starch synthesis due to the plant’s inability to initiate starch
granules in most of the cells. The block in starch synthesis was found to result in an unprecedented accumulation of ADP-Glc, thereby sequestering much of
the plastidial adenine nucleotide in a metabolically
inaccessible form. This will severely limit the synthesis
and availability of ATP for photosynthetic CO2 fixation
and many other processes and will also trigger ROS
production, leading to photooxidative damage of the
photosynthetic apparatus within the chloroplasts. Restricting the synthesis of ADP-Glc in the chloroplasts by introducing the pgm1 or aps1 mutation into ss3/ss4 greatly
reduced the accumulation of ADP-Glc and restored both
the photosynthetic capacity and growth of the plants.
This confirmed that the massive accumulation of ADPGlc in the ss3/ss4 mutant underlies the chlorotic and
stunted growth phenotype of this mutant and explains
why it differs so markedly from other starch-deficient
mutants.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
All Arabidopsis (Arabidopsis thaliana) mutants were in the Columbia-0 (Col-0)
background. The ss3, ss4, and ss3/ss4 mutants were as described by Szydlowski
et al. (2009), and the pgm1 mutant was from Caspar et al. (1985). The aps1
mutant (SALK_ 040155) contains a transfer DNA insertion in the APS1 gene
(At5g48300; Alonso et al., 2003), and homozygous knockout plants were selected
using PCR-based genotyping. Lines carrying triple mutations were obtained by
crossing and selecting homozygous triple mutant plants from the segregating F2
populations using PCR-based genotyping. All primers used are described in
Supplemental Table S1.
Unless otherwise specified, plants were grown in growth cabinets at 23°C
(day)/20°C (night), 70% humidity, and an irradiance of 120 mE m22 s21 supplied by white fluorescent lamps. Seeds were sown in soil and irrigated with
0.53 Murashige and Skoog medium (Murashige and Skoog, 1962).
Photosynthesis Measurements
Photosynthetic gas exchange was measured using a portable infrared gas
analyzer (model LI-6400; LI-COR Biosciences), which allows environmental
conditions inside a standard leaf clamp of the chamber to be precisely controlled.
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Ragel et al.
Air temperature in the chamber was set at 25°C, and the relative humidity was
maintained at 50%. Light-response curves were determined on one of the upper
leaves of the plants, progressively increasing the irradiance from 0 to 50 and
then to 100, 250, 500, 750, 1,000, 1,500, and 2,000 mmol quanta m22 s21 in
stepwise changes every 3 min. The CO2 assimilation rate was determined on
3-week-old plants grown in a controlled environment under LD conditions (16 h
of light/8 h of dark) with an irradiance of 100 mmol quanta m22 s21 at a constant
22°C temperature.
Photosyn Assistant software, developed by Dundee Scientific (R. Parsons
and S.A. Ogston), has been used to determine parameters such as CO2 assimilation rates to help in the comparison between the mutants.
Gas exchange of whole Arabidopsis rosettes was measured with a custombuilt multichamber system connected in parallel to an infrared gas analyzer
(Li-7000; LI-COR). Individual plants were introduced into each of eight chambers, and after an adaptation period of 2 d, gas exchange was measured for a
complete 24-h light/dark period. During the measurement, air with 380 mL L21
CO2 and a relative humidity of 60% was directed through the system. Each
chamber was measured consecutively for 6 min, and the average value was
taken for the light period. At the end of the experiment, a photograph was taken
to calculate the projected leaf area for each plant. Based on the area, photosynthetic and respiration rates were calculated with the ΔCO2 and ΔH2O values
gained from the gas-exchange system.
Chlorophyll fluorescence emission parameters were measured at 22°C with
a PAM 2000 fluorometer (Walz). The Fv/Fm was calculated from the measured
parameters using the following equation: Fv/Fm = (Fm 2 Fo)/Fm, where Fo is
the initial minimal fluorescence emitted from leaves dark adapted for 1 h and
Fm is the maximal fluorescence elicited by saturating actinic light. The FPSII
was calculated from the measured parameters using the following equation:
FPSII = Fm9 2 F9/Fm9, where Fm9 is the maximal fluorescence from lightadapted leaves and F9 is the fluorescence emission from light-adapted leaves.
Plant Growth Measurement
Plant growth was determined by weighing the aerial part of the plants.
In the case of pgm1 and pgm1/ss3/ss4 mutant plants, the plant growth was
measured based on whole rosette area. Digital images were taken every 2 or
3 d at the same time. The digital images were converted with Photoshop
(Adobe Photoshop CS4 Extended; Adobe Systems) to black-and-white images,
with the rosette being black and the background being white. ImageJ 1.42q
(National Institutes of Health) was used to calculate the leaf area, based on the
scale, from the black-and-white images.
Analytical Methods
Seven discs of rosette leaves (5–10 mg) from independent plants were used
for the determination of chlorophylls a and b and carotenoids. Pigments were
extracted with methanol and quantified according to the methods described
by Porra et al. (1989) for chlorophylls and by Lichtenthaler and Buschmann
(2001) for carotenoids. The extraction and quantitation of anthocyanins were
performed as described (Rabino and Mancinelli, 1986) using seven rosette
leaves from independent plants. The concentration of MAD in plants was
determined using the thiobarbituric acid method described by Buege and Aust
(1978). Soluble sugars and starch were measured enzymatically in ethanolic
extracts and the ethanol-insoluble residue as described by Hendriks et al.
(2003). ADP-Glc, phosphorylated intermediates, and organic acids were measured in chloroform-methanol extracts using high-pressure anion-exchange
chromatography coupled to MS/MS as described (Lunn et al., 2006). ATP,
ADP, and AMP were measured enzymatically in freshly prepared TCA extracts
as described (Weiner et al., 1987; Trethewey et al., 1998).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Determination of levels of phosphorylated intermediates and organic acids of wild-type, ss3, ss4, ss3/ss4, aps1, pgm1,
aps1/ss3/ss4, and pgm1/ss3/ss4 Arabidopsis plants grown in 16-h-light/
8-h-dark conditions (20°C/18°C, irradiance of 150 mE m22 s21) and harvested at 8 h into the light period.
Supplemental Figure S2. Growth rates of Col-0, aps1/ss3/ss4, pgm1/ss3/ss4,
and their parental lines.
Supplemental Figure S3. Total adenine nucleotide pool size (ATP + ADP +
AMP + ADP-Glc) was measured in wild-type, ss3, ss4, ss3/ss4, aps1, aps1/
ss3/ss4, pgm, and pgm/ss3/ss4 Arabidopsis plants grown in 16-h-light/
8-h-dark conditions (20°C/18°C, irradiance at 150 mE m22 s21) and harvested at 8 h into the light period.
Supplemental Table S1. Primers used for the identification of the triple
mutant plants.
Received June 17, 2013; accepted July 18, 2013; published July 21, 2013.
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