Journal of Experimental Botany, Vol. 56, No. 415, pp. 1297–1303, May 2005
doi:10.1093/jxb/eri130 Advance Access publication 14 March, 2005
This paper is available online free of all access charges (see http://jxb.oupjournals.org/open_access.html for further details)
RESEARCH PAPER
The sources of carbon and reducing power for fatty acid
synthesis in the heterotrophic plastids of developing
sunflower (Helianthus annuus L.) embryos
Rafael Pleite2, Marilyn J. Pike1, Rafael Garcés2, Enrique Martı́nez-Force2 and Stephen Rawsthorne1,*
1
2
Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
Instituto de la Grasa, CSIC, Avd. Padre Garcı´a Tejero, 4. 41012, Seville, Spain
Received 29 September 2004; Accepted 2 February 2005
Abstract
The provision of carbon substrates and reducing power
for fatty acid synthesis in the heterotrophic plastids of
developing embryos of sunflower (Helianthus annuus L.)
has been investigated. Profiles of oil and storage protein
accumulation were determined and embryos at 17 and
24 days after anthesis (DAA) were selected to represent
early and late periods of oil accumulation. Plastids
isolated from either 17 or 24 DAA embryos did not
incorporate label from [1-14C]glucose 6-phosphate
(Glc6P) into fatty acids. Malate, when supplied alone,
supported the highest rates of fatty acid synthesis by the
isolated plastids at both stages. Pyruvate supported
rates of fatty acid synthesis at 17 DAA that were comparable to those supported by malate, but only when
incubations also included Glc6P. The stimulatory effect
of Glc6P on pyruvate utilization at 17 DAA was related to
the rapid utilization of Glc6P through the oxidative
pentose phosphate pathway (OPPP) at this stage. Addition of pyruvate to incubations containing [1-14C]Glc6P
increased OPPP activity (measured as 14CO2 release),
while the addition of malate suppressed it. Observations
of the interactions between the rate of metabolite utilization for fatty acid synthesis and the rate of the OPPP
are consistent with regulation of the OPPP by redox
control of the plastidial glucose 6-phosphate dehydrogenase activity through the demand for NADPH. During
pyruvate utilization for fatty acid synthesis, flux through
the OPPP increases as NADPH is consumed, whereas
during malate utilization, in which NADPH is produced
by NADP-malic enzyme, flux through the OPPP is
decreased.
Key words: Embryo, fatty acid synthesis, Helianthus annuus,
oxidative pentose phosphate pathway, plastid, starch.
Introduction
In plants, de novo fatty acid synthesis occurs in plastids. The
first committed step of fatty acid synthesis is the formation of
malonyl-CoA from acetyl-CoA and bicarbonate which is
catalysed by acetyl-CoA carboxylase (Harwood, 1988).
Since acetyl-CoA cannot cross the plastid membrane, it
must be generated within the plastid using precursors which
are synthesized inside the plastid or actively imported from
the cytosol. There is a broad knowledge about the nature of
the precursors used for fatty acid biosynthesis by plastids
isolated from different plant species (Rawsthorne, 2002).
The imported precursors include glucose-6-phosphate
(Glc6P), dihydroxyacetone phosphate, phosphoenolpyruvate, pyruvate, malate, and acetate, and their relative rates
of utilization depend upon the plant species, the tissue
studied, and also the developmental stage. For example,
Glc6P and pyruvate were the metabolites that supported the
highest rates of fatty acid synthesis by plastids isolated from
developing Brassica napus embryos at the early- and midoil stages of development, respectively (Eastmond and
Rawsthorne, 2000). By contrast, malate supported high
rates of fatty acid biosynthesis by plastids isolated from
developing castor bean (Ricinus communis L.) endosperm
(Smith et al., 1992).
In addition to a carbon supply, the production of fatty
acids requires the provision of reducing power, in the form of
NADPH and NADH (Slabas and Fawcett, 1992). The source
of reductant for fatty acid synthesis in non-photosynthetic
tissues is unclear. The available data for heterotrophic
* To whom correspondence should be addressed. Fax: +44 (0)1603 450014. E-mail: [email protected]
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1298 Pleite et al.
plastids point to the generation of reductant during the
synthesis of acetyl-CoA from Glc6P, malate or pyruvate
(Smith et al., 1992; Kang and Rawsthorne, 1996), or via the
plastidial oxidative pentose phosphate pathway (OPPP)
(Dennis, 1989; Kang and Rawsthorne, 1996; Eastmond
and Rawsthorne, 2000; Schwender et al., 2003). However,
much of these data are derived from the study of B. napus
embryos which have plastids that have both photosynthetic
and heterotrophic properties (Eastmond et al., 1996; Eastmond and Rawsthorne, 1998).
Developing sunflower (Helianthus annuus) embryos are
achlorophyllous and therefore incapable of generating reductant via photosynthesis, unlike the developing embryos
of B. napus (Eastmond et al., 1996; Schwender et al., 2003).
We have therefore studied which carbon precursors can be
used by the heterotrophic plastids from sunflower embryos
for the generation of the acetyl-CoA and reducing power that
are required for de novo fatty acid synthesis.
Materials and methods
Plant material, chemicals, and reagents
Sunflower (Helianthus annuus L.) plants (public cultivar RHA-274)
were grown in a glasshouse, with supplementary illumination from
sodium lamps giving a 16 h day length providing a photon flux
density of 500 lmol mÿ2 sÿ1, at average day/night temperatures of 27 8C
and 19 8C, respectively, and fed with Phostrogen fertilizer as
recommended by the manufacturer (pbi Home & Garden Limited,
Waltham Cross, Herts., UK). The outermost two rows of florets on
the flower head opened and anthesed on the same day. Seeds were
collected from these two rows of the flower head only and at defined
days after anthesis (DAA).
Substrates, cofactors and coupling enzymes were purchased from
either Sigma Chemical Company (Poole, Dorset, UK) or Boehringer
Mannheim (Lewes, Sussex, UK). Radioisotopes were obtained from
Amersham International (Amersham, Bucks, UK) and NEN DuPont
(Herts, UK). Water was from an ELGA water purification system
(ELGA Process Water, High Wycombe, Bucks, UK).
Determination of oil, protein, and starch content of embryos
Oil content of the embryos was measured according to Kochhar and
Rossell (1987). Four grams of embryos (achenes without hull and
seed coat) were homogenized and oil was extracted in boiling
petroleum ether.
Protein content in total homogenates of embryos was measured
according to Bradford (1976) using Bio-Rad Protein Assay Dye
Reagent with bovine serum albumin (BSA) as a standard. Three
sunflower embryos were homogenized in 1 ml of 50 mM HEPES/
NaOH (pH 7.4) using a glass homogenizer and the homogenate was
centrifuged at 16 000 g for 20 min before assay.
Starch was extracted and measured according to Smith (1988)
using five sunflower embryos.
Isolation of plastids from developing embryos
Plastids were isolated according to Kang and Rawsthorne (1994) with
slight modifications. Isolated embryos were kept on ice in a modified
plastid isolation medium (PIM: 0.5 M sorbitol, 20 mM 4-(2hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES)/NaOH
(pH 7.4), 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol (DTT), and 1% (w/v) BSA for up to 2 h before experiments.
The embryos were homogenized by chopping using a razor blade.
Plastids were washed and resuspended in PIM without BSA.
The intactness of the plastid preparations was determined by the
latency of enolase activity, as described by Smith et al. (1992).
Pyrophosphate-dependent phosphofructokinase activity (PFP) and
NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH)
were used as cytosolic and plastid markers, respectively, according to
Kang and Rawsthorne (1994).
Measurement of starch synthesis and OPPP activity
in isolated plastids
The incorporation of 14C from [1-14C]glucose 6-phosphate (Glc6P)
into starch (methanol/KCl insoluble material, verified as starch
according to Hill and Smith (1991)) and release of 14CO2 via the
oxidative pentose phosphate pathway (OPPP) were measured simultaneously as described by Kang and Rawsthorne (1996).
Fatty acid synthesis by isolated plastids
The incorporation of carbon from metabolites into fatty acids was
determined as described by Kang and Rawsthorne (1994). Incubations
were conducted in a 0.15 ml reaction mixture containing PIM buffer, 6
mM MgCl2, 4 mM ATP, 10 mM NaHCO3, 0.5 mM NAD, 0.2 mM
CoA, 0.2 mM thiamine PPi, and one of the following substrates:
[1-14C]Glc6P (18 GBq molÿ1), [2-14C]acetate, [U-14C]malic acid, and
[2-14C]pyruvic acid. The specific radioactivity of the 14C-labelled
acetate, malate, and pyruvate was between 5 and 54 GBq molÿ1.
Reactions were initiated by the addition of 25 ll of washed plastids.
Incubations were carried out for 60 min at 25 8C with gentle mixing.
Reactions were stopped by addition of 67 ll 4 M methanolic NaOH.
Incorporation into fatty acid was measured as 14C partitioned into the
organic phase according to Fox et al. (2001). Incubations were
saponified by heating at 80 8C for 1 h, and then extracted with 33 ll
of 12 M HCl, 200 ll of saturated NaCl solution, and 500 ll of isohexane. The purified upper organic fraction was dried under a stream of
nitrogen, resuspended in oleic acid in hexane (1 mg mlÿ1; w/v) and the
14
C products were resolved by TLC on a silica gel G plate (Analtech,
Luton, UK) using a solvent system for neutral lipids (Mudd and
Dezacks, 1981). The band containing the free fatty acid fraction was
scraped from the plate and the radioactivity determined by scintillation
counting. The results are expressed as the differences in label in the free
fatty acid fraction between incubations stopped after 60 min and at
zero time.
Results
Isolation of plastids from developing sunflower
embryos
To establish the stages of seed development at which to
isolate plastids for use in studies of fatty acid synthesis, the
accumulation of storage products in the embryos was
determined (Fig. 1). Oil accumulation was essentially linear
between 12 and 25 DAA and then ceased. The main period
of protein deposition was between 15 and 25 DAA but
continued up to 31 DAA. Starch content was less than 1.6%
of the combined total protein and oil content during seed
development (data not shown). To investigate whether their
metabolism changed during development, plastids were
isolated from embryos at 17 and 24 DAA, representing early
and late periods of oil accumulation. During plastid isolations, cytosolic and plastidial marker enzymatic activities
were measured to assess the purity of plastid preparations.
Fatty acid carbon source in sunflower 1299
Less than 0.5% of the total PFP activity (cytosolic marker)
in the original homogenate was recovered in the washed
plastid preparations. For NADP-GAPDH, the plastid marker,
an average of 11% of the total activity in the original
homogenate was recovered in the same preparations.
Plastid integrity was measured using the latency of enolase
activity. For all plastid preparations, intactness was between 75–90%. With regard to these properties, the washed
plastid fractions from sunflower embryos were similar to
those for plastids isolated from castor bean endosperm
(Smith et al., 1992; Eastmond et al., 1997).
manner at both stages and rates of incorporation of all
substrates were linear over a 60 min period. Glc6P was not
used as a carbon source for fatty acid synthesis at either
stage (data not shown). At both developmental stages, the
highest rate of fatty acid synthesis was supported by malate
(Fig. 3). At 17 DAA, plastids utilized malate as a carbon
source for fatty acid synthesis at a rate that was 5-fold
greater than that for pyruvate and 15-fold that for acetate.
Fatty acid synthesis by isolated plastids
It was investigated which metabolites could be used as
carbon sources for fatty acid synthesis by plastids isolated
from 17 DAA sunflower embryos and how this was
affected by the concentration of the supplied metabolite
(Fig. 2). The rate of incorporation of carbon from malate
and pyruvate into fatty acids was saturated at 3 mM and
1 mM, respectively. Very similar results were found for
leucoplasts isolated from castor bean endosperm, where the
rate of fatty acid synthesis became substrate-saturated at
3–5 mM for malate and 1 mM for pyruvate (Smith et al.,
1992). Isolated plastids supplied with [1-14C]Glc6P at up to
5 mM did not synthesize any detectable labelled fatty acids
(data not shown). Incorporation of acetate was linearly
dependent on the acetate concentration, up to the maximum
of 5 mM that was used here.
To determine if the utilization of metabolites for fatty
acid synthesis changed during development, plastids isolated from 17 or 24 DAA sunflower embryos were incubated with 3 mM 14C-labelled acetate, Glc6P, malate, or
pyruvate. Carbon from malate and pyruvate was incorporated into fatty acids in an ATP- and intactness-dependent
Fig. 1. Accumulation of lipid (closed dircles) and protein (open circles)
during development of sunflower seeds. Each data point represents the
mean 6SE of measurements made on three separate batches of seeds.
Fig. 2. Concentration dependence of the incorporation of carbon from
metabolites into fatty acids by isolated plastids. Rates of incorporation of
carbon from (closed circles) malate, (open circles) pyruvate, and (closed
inverted triangles) acetate are expressed on an acetate-equivalent basis
and each value is the mean 6SE of measurements made on three separate
plastid preparations from seeds at 17 DAA.
Fig. 3. Developmental changes in the incorporation of carbon from
metabolites into fatty acids by isolated plastids. Plastids were prepared
from developing seeds at 17 DAA (solid bars) or 24 DAA (shaded bars)
incubated with 3 mM 14C-labelled metabolites and the incorporation of
carbon into fatty acids was determined. Rates of incorporation of carbon
from malate, pyruvate, and acetate are expressed on an acetate-equivalent
basis and each value is the mean 6SE of measurements made on three
separate plastid preparations.
1300 Pleite et al.
At 24 DAA, the rate of fatty acid synthesis when malate
was supplied was 12-fold greater than that for pyruvate and
no incorporation of label was detectable from acetate. The
in vitro rates of fatty acid synthesis by isolated plastids were
substantially lower at the later stage of seed development
(Fig. 3). Rates at 24 DAA were 54% and 82% lower than at
17 DAA for malate and pyruvate, respectively.
Glucose 6-phosphate metabolism by isolated plastids
As Glc6P was not used as a carbon source for fatty acid
biosynthesis, it was investigated whether it could cross the
plastid membrane and be used as a carbon source for other
metabolic pathways like starch synthesis or the production of reducing power through the OPPP (Kang and
Rawsthorne, 1996). Incorporation of label from [114C]Glc6P
into starch and CO2 was dependent on the intactness of the
plastids, and starch synthesis was also ATP-dependent. The
rate of Glc6P utilization for both was linear over a 60 min
incubation (data not shown). The concentration dependence
of the incorporation of Glc6P into starch (methanol/KClinsoluble material) was determined for plastids from 17
DAA embryos. The rate of incorporation was saturated at
a Glc6P concentration of about 0.5 mM (data not shown).
The partitioning of Glc6P between starch synthesis and
the OPPP was studied in plastids from 17 and 24 DAA
sunflower embryos (Table 1). When expressed on a per unit
hexose basis, the utilization of Glc6P for starch synthesis
was about twice that metabolized through the OPPP at both
developmental stages. The fluxes of Glc6P into starch
synthesis and through the OPPP were decreased by about
85% between 17 and 24 DAA. The release of 14CO2 from
[1-14C] Glc6P in the absence of incorporation of label into
fatty acids implies that a sink for reducing power is
operative in the isolated plastids, but this cannot be fatty
acid synthesis supplied by glycolysis as carbon from Glc6P
was not utilised for fatty acid synthesis by these plastids. In
order to investigate the interaction between OPPP activity
and the demand for NADPH by fatty acid synthesis, it was
decided to study how OPPP activity was affected by
changing the rate of fatty acid synthesis.
Interactions between fatty acid synthesis and the
OPPP during metabolism by isolated plastids
To investigate interactions between fatty acid synthesis and
the OPPP, plastids were isolated from 17 and 24 DAA
sunflower embryos and the incorporation of carbon from
14
C-malate or 14C-pyruvate into fatty acids was determined
in the presence or absence of unlabelled 1 mM Glc6P (Fig. 4).
There was no significant change (t-test, P <0.05) in the rate
of fatty acid synthesis from malate when Glc6P was supplied
at either developmental stage. However, Glc6P stimulated
the rate of incorporation of carbon from pyruvate into fatty
acids by plastids isolated from 17 DAA embryos by more
than 4-fold to give a rate that was not significantly different
(P <0.05) from that attained in the incubations with malate
(Fig. 4). There was no significant stimulation of incorporation of carbon from pyruvate into fatty acids by
Glc6P in incubations with plastids isolated from 24 DAA
embryos. As Glc6P was not used as the carbon source for
fatty acid biosynthesis when supplied separately (see above),
it was concluded that the stimulation of pyruvate-dependent
fatty acid synthesis by Glc6P must be due to the production
of NADPH through the OPPP. This cofactor, required for
de novo fatty acid synthesis, is not produced in the plastid
when pyruvate is metabolized via the pyruvate dehydrogenase complex, but is produced via plastidial NADP-malic
enzyme when malate is supplied.
To test whether the OPPP activity was affected by fatty
acid synthesis, plastids were isolated from 17 and 24 DAA
sunflower embryos and incubated with [1-14C]Glc6P alone
and with unlabelled malate or pyruvate. The label incorporated into CO2 was then measured (Fig. 5). When
Table 1. Developmental changes in the metabolism of Glc6P by
isolated plastids
Plastids were isolated from 17 DAA or 24 DAA seeds and incubated with
1 mM [1-14C]Glc6P. Starch synthesis was measured as incorporation of
label into a methanol/KCl insoluble fraction and the OPPP was measured
as 14CO2 released during the incubation. Rates are expressed on a mole of
Glc6P basis and each value is the mean 6SE of measurements made on
three separate plastid preparations.
Pathway
Age of seed (DAA)
17
24
ÿ1
Starch synthesis
OPPP
(nmol Glc6P h
13.062.2
5.461.4
ÿ1
mg
protein)
1.760.6
0.960.1
Fig. 4. Effect of Glc6P on fatty acid synthesis by isolated plastids.
Incorporation of carbon from 3 mM 14C-labelled malate or pyruvate into
fatty acids by plastids isolated from seeds at 17 or 24 DAA was measured
in the presence (shaded bars) or absence (solid bars) of 1 mM unlabelled
Glc6P. Rates of incorporation of carbon are expressed on an acetateequivalent basis and each value is the mean 6SE of measurements made
on three separate plastid preparations.
Fatty acid carbon source in sunflower 1301
Fig. 5. Effect of fatty acid synthesis on Glc6P metabolism by isolated
plastids. Plastids were isolated from 17 (solid bars) or 24 (shaded bars)
DAA seeds and incubated with 1 mM [1-14C]Glc6P alone or together
with 3 mM unlabelled malate (Mal) or pyruvate (Pyr). OPPP activity was
measured as 14CO2 released during a 60 min incubation. Rates of 14CO2
release are expressed on a mole of Glc6P basis and each value is the mean
6SE of measurements made on three separate plastid preparations.
pyruvate was added to incubations, the rate of 14CO2
release from Glc6P was stimulated by 3.0-fold and 2.4-fold
for plastids isolated from 17 and 24 DAA embryos,
respectively. By contrast, when malate was added, the
rate of 14CO2 release at both stages decreased by an average
of 83% at both developmental stages.
Discussion
The data presented here (i) reveal that malate and pyruvate
represent the most likely metabolites for uptake and incorporation into fatty acids during oil synthesis in developing embryos of sunflower, (ii) show that the reducing
power for fatty acid synthesis can be produced through
metabolism of malate through NADP-malic enzyme and
PDC, and/or metabolism of Glc6P through the OPPP, and
(iii) provide a model for fatty acid synthesis in this nonphotosynthetic storage tissue.
When carbon sources were supplied alone to plastids
isolated from developing sunflower embryos, malate supported the highest rates of fatty acid synthesis throughout the
period of oil accumulation. However, pyruvate could support rates of fatty acid synthesis at 17 DAA (i.e. early oil
accumulation) that were comparable to those of malate when
incubations also included Glc6P, which led to a stimulation
in the rate of incorporation of carbon from pyruvate into fatty
acids. It is proposed here that, in isolated plastids, this
stimulation by Glc6P is due to the production of NADPH by
the operation of the OPPP. The principal evidence that supports this proposal is that addition of unlabelled pyruvate to
plastids, under incubation conditions that enable fatty acid
synthesis, leads to a large stimulation of 14CO2 release from
[1-14C]Glc6P; i.e. the activity of the OPPP is increased by
a demand for NADPH created through fatty acid synthesis
(Slabas and Fawcett, 1992). This is because when pyruvate
is the sole supplied metabolite, acetyl-CoA and NADH are
generated by the plastidial pyruvate dehydrogenase complex
(PDC), but NADPH is not. As shown here for plastids from
developing sunflower embryos, malate, when provided as
the sole metabolite, also facilitated higher rates of fatty acid
synthesis by plastids isolated from castor endosperm than
when pyruvate was provided (Smith et al., 1992). This
ability of castor endosperm leucoplasts preferentially to
utilize malate has been explained, in part, by the activity of
a malate transporter on the plastid envelope (Eastmond et al.,
1997). However, although the leucoplasts of castor endosperm contain the enzymes of the OPPP (Dennis, 1989), the
ability of Glc6P to stimulate the incorporation of carbon
from pyruvate into fatty acids has not been studied. There is
clear evidence that Glc6P metabolism through the OPPP
stimulates the incorporation of carbon from pyruvate into
fatty acids by plastids isolated from developing embryos of
B. napus (Kang and Rawsthorne, 1996; Johnson et al., 2000)
and that the OPPP can provide at least part of the reducing
power for fatty acid synthesis in whole B. napus embryos
(Schwender et al., 2003).
This study also reveals that the interaction between
utilization of carbon substrates for the synthesis of acetylCoA and for the production of reducing power is likely to be
mediated through the redox poise of the oxidative steps of
the OPPP which is controlled by Glc6P dehydrogenase
(Kruger and von Schaewen, 2003). As described above,
pyruvate utilization for fatty acid synthesis by plastids from
sunflower embryos is stimulated by the addition of Glc6P. In
contrast, Glc6P addition has no effect on the utilization of
malate. Furthermore, while addition of pyruvate stimulated
the activity of the OPPP, malate suppressed the OPPP
activity to a level below that which occurred in the absence
of a sink created by fatty acid synthesis; i.e. when Glc6P was
provided alone and was metabolized to starch and through
the OPPP (but not to fatty acids). It is proposed that when
malate is provided to sunflower embryo plastids, the
activities of plastidial NADP-malic enzyme and PDC lead
to stoichiometric provision of NADPH, NADH and acetylCoA, as described previously for castor leucoplasts (Smith
et al., 1992). Under these conditions there would be no
demand for additional NADPH from the OPPP and utilization of malate should be unaffected by Glc6P. Not only was
this seen, but also the suppression of OPPP activity by
malate oxidation, which suggests that the generation of
NADPH by NADP-malic enzyme was sufficient to modulate Glc6PDH activity through redox status in the plastid
(Kruger and von Schaewen, 2003) and to reduce flux
through the oxidative steps of the OPPP.
The efficient utilization of malate for fatty acid synthesis
by the achlorophyllous plastids from sunflower embryos
1302 Pleite et al.
and castor endosperm (Smith et al., 1992) is in contrast to
its relatively poor utilization by chlorophyllous plastids of
B. napus (Kang and Rawsthorne, 1994) and Linum usitatissimum (Troufflard et al., 2004) embryos. This is not
simply explained by a lack of plastidial NADP-malic
enzyme in the plastids of chlorophyllous storage tissues
because plastids from B. napus embryos contain activities
of this enzyme in excess of the rate of fatty acid synthesis
(Kang and Rawsthorne, 1994). It is therefore possible that
photosynthetic electron transport is used to provide a proportion of the reducing power for fatty acid synthesis in
chlorophyllous plastids from storage organs (Schwender
et al., 2003). On the other hand, in the absence of
chlorophyll, the stoichiometry of the carbon skeleton and
reducing power production can be maintained through
oxidative metabolism of malate, supported by a malate
transporter (Eastmond et al., 1997). Whether this observation holds true for fatty acid synthesis in a wider range of
chlorophyllous and achlorophyllous tissues that store oil
remains to be investigated.
These studies of Glc6P metabolism by sunflower embryo
plastids revealed that the OPPP is active in the absence of
other metabolites that generate a sink for reductant. A similar
observation has been made for plastids isolated from
B. napus embryos (Hutchings et al., 2005). These observations contrast with measurements of OPPP activity in
plastids isolated from roots of Pisum sativum in which the
activity of the OPPP is negligible in the absence of
metabolites that generate a sink for reductant during nitrogen
assimilation (Bowsher et al., 1992). The nature of the
endogenous sink for reductant in the plastids isolated from
developing embryos of sunflower and B. napus remains to be
elucidated, but its presence makes quantitative comparisons
between the fluxes of carbon to fatty acids and reductant
utilization impossible to determine.
It was notable that although sunflower embryos accumulated only tiny quantities of starch in comparison to oil
throughout their development, plastids isolated from them
utilized Glc6P for starch synthesis at rates that were at least
comparable, on a per mole of substrate utilized, to those of
fatty acid synthesis and the OPPP at both 17 and 24 DAA.
This observation implies that starch may be turned over in
sunflower embryos, as it is in embryos of other oilseeds.
For example, starch accumulates and is subsequently lost
during seed development in B. napus and arabidopsis (da
Silva et al., 1997; Baud et al., 2002), and plastids isolated
from B. napus embryos that are in the period of net starch
degradation synthesize starch at high rates (Eastmond and
Rawsthorne, 2000). On a shorter timescale, developing
embryos of arabidopsis that contain starch can turn it over
on a diurnal basis depending on the growth conditions
(Schreck, 2002). Troufflard et al. (2004) have also reported
that plastids isolated from linseed embryos are capable of
rates of starch synthesis in vitro that far exceed the rate of
starch accumulation in vivo.
Another feature of Glc6P metabolism by sunflower
plastids was that it was barely utilized as a carbon source
for fatty acid synthesis. Utilization of Glc6P for fatty acid
synthesis by isolated castor leucoplasts is also very poor
compared with that from malate (Miernyk and Dennis,
1983), although plastids isolated from developing B. napus
embryos readily utilize Glc6P for fatty acid synthesis
compared with pyruvate, phosphoenolpyruvate, malate,
and acetate (Kang and Rawsthorne, 1994; Eastmond and
Rawsthorne, 2000; Kubis et al., 2004). The observations for
sunflower and castor, and for plastids from a range of
species/tissues, are probably due to low or negligible activities of certain glycolytic enzymes within the plastid (see
Rawsthorne, 2002, and references therein). The plastids
from B. napus embryos, in contrast, have been shown to
have glycolytic enzyme activities that are sufficient to
support the rates of lipid synthesis that are observed
in vivo (Eastmond and Rawsthorne, 2000). An alternative
explanation for the poor incorporation of Glc6P into fatty
acids could be that the carbon is metabolized largely through
the oxidative pentose phosphate pathway before being
utilized for fatty acid synthesis. This would be undetectable
with the use of [1-14C]Glc6P as 14CO2 would be released
from the 1-C position in the oxidative steps. While this
cannot be ruled out, Schwender et al. (2004) have provided
evidence, based on 13C-labelling studies with isolated
chlorophyllous oilseed embryos, for a pentose phosphate
‘shunt’ for Glc6P that provides carbon for fatty acid
synthesis. This pathway only utilizes the non-oxidative
steps of the pentose phosphate pathway and Rubisco, and if
it were operating in sunflower this would be detected
through the incorporation of carbon from [1-14C]Glc6P
into fatty acids because label in the 1-C position would be
conserved. Schwender et al. (2004) reported that there was
no evidence for the operation of this novel pathway in
isolated, developing sunflower embryos in culture, and the
data for isolated plastids from this study also suggest that it is
not functional to any major extent.
Acknowledgements
We are grateful to Drs Kay Denyer, Matthew Hills, and Stanislav
Kopriva for their helpful comments on the manuscript. Mr Ian
Hagon and his team are thanked for horticultural support. This work
was supported by the Biotechnology and Biological Sciences Research Council by the Competitive Strategic Grant to JIC (SR and
MJP), by the Consejo Superior de Investigaciones Cientificas (RG
and EM-F), and by funding from the Programa Nacional de
Formación de Profesorado Universitario (Ministerio de Educación
y Ciencia) (RP).
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