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Plant Physiol. (1999) 120: 401-410
Expression of the Granule-Bound Starch Synthase I
(Waxy) Gene from Snapdragon Is Developmentally
and
Circadian Clock Regulated1
Angel Mérida,
José M. Rodríguez-Galán2,
Coral Vincent, and
José M. Romero*
Instituto de Bioquímica Vegetal y Fotosíntesis,
Centro de Investigaciones Científicas Isla de la Cartuja,
C/Americo Vespucio s/n, 41092 Seville, Spain (A.M., J.M.R.-G., J.M.R.); and John Innes Centre, Norwich NR4 7UH, United Kingdom
(C.V.)
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ABSTRACT |
The
granule-bound starch synthase I (GBSSI or waxy) enzyme catalyzes one of
the enzymatic steps of starch synthesis. This enzyme is responsible for
the synthesis of amylose and is also involved in building the final
structure of amylopectin. Little is known about expression of GBSSI
genes in tissues other than storage organs, such as seeds, endosperm,
and tuber. We have isolated a gene encoding the GBSSI from snapdragon
(Antirrhinum majus). This gene is present as a single
copy in the snapdragon genome. There is a precise spatial and
developmental regulation of its expression in flowers.
GBSSI expression was observed in all floral whorls at
early developmental stages, but it was restricted to carpel before
anthesis. These results give new insights into the role of starch in
later reproductive events such as seed filling. In leaves the mRNA
level of GBSSI is regulated by an endogenous circadian
clock, indicating that the transition from day to night may be
accompanied by abolition of expression of starch synthesis genes. This
mechanism does not operate in sink tissues such as roots when grown in
the dark.
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INTRODUCTION |
Starch is the most important form of C reserve in plants.
Long-term storage starch is found in storage organs such as tubers, endosperm, embryos, or storage roots, and transitory starch is present
in photosynthetically active tissues such as leaves. The starch
biosynthesis pathway has been extensively studied in diverse species
such as maize, potato, pea, and others (for review, see Martin and
Smith, 1995 ; Nelson and Pan, 1995; Preiss and Sivak, 1996; Smith et
al., 1997), and considerable progress has been made toward
understanding the role of each enzymatic step needed to build the final
structure of the starch granule (Ball et al., 1996; Buleon et al.,
1997). These studies have demonstrated that plants use different
isoforms for each enzyme of the biosynthetic pathway: ADP-Glc
pyrophosphorylase, starch synthases, and starch-branching enzyme, and
each isoform is encoded by a different gene. For example, three
isoforms for starch-branching enzyme have been found in maize (Boyer
and Preiss, 1981), and the genes encoding the three isoforms have been
previously described (Baba et al., 1991; Fisher et al., 1995; Gao et
al., 1997). Three genes encoding for the large subunit of the
ADP-Glc pyrophosphorylase have been cloned in
Arabidopsis (Villand et al., 1993). In potato three genes encoding distinct isoforms of the starch synthase have been previously described
(Van der Steege et al., 1992; Edwards et al., 1995; Marshall et al.,
1996; Abel et al., 1997).
One of these isoforms, GBSSI, is located exclusively in the starch
granule (Sivak et al., 1993). It was originally identified in maize as
the product of the waxy gene, and through mutant analysis it
has been shown to be responsible for the synthesis of linear glucan
(amylose) in starch (Nelson and Pan, 1995). Genes encoding the
orthologous protein have been isolated from many different plant
species such as potato (Van der Steege et al., 1992), pea (Dry et al.,
1992), barley (Rohde et al., 1988), and wheat (Clark et al., 1991).
Analysis of mutants in rice, Amaranthus, potato, and pea has
demonstrated that GBSSI is also responsible for the synthesis of amylose in storage organs of these plants (Smith et al.,
1997, and refs. therein). However, on the basis of biochemical and
mutant studies, the existence of different isoforms responsible for the
GBSSI activity in different organs has been proposed for pea (Denyer et
al., 1997), rice (Taira et al., 1991), and wheat (Nakamura et al.,
1998).
Most of the studies of starch biosynthesis have been carried out on
storage organs such as tuber or endosperm because of the economic
importance of the long-term reserve form of starch and the relative
availability of both enzymes and product. There is much less
information available about starch synthesis in other organs and
tissues of the plant, although changes in the synthesis and
mobilization of transitory starch affect processes such as growth rate,
flowering time, and seed filling (Bernier et al., 1993; Schulze et al.,
1994). It has been proposed that mobilization of starch stored in
leaves and stems to Suc provides one of the early signals for the
induction of flowering (Bernier et al., 1993). Analysis of the growth
of a starch-less mutant of Arabidopsis (deficient in plastidial
phosphoglucomutase activity; Caspar et al., 1985) has shown that
synthesis of starch is necessary not only to maintain normal growth
rates under a natural day/night regime but also to promote other
developmental changes such as flowering or seed filling (Schulze et
al., 1994).
The main characteristic of transitory starch is that it is synthesized
during the day and degraded during the night to supply the C
requirements of the plant. A circadian regulation of starch accumulation during the day has been proposed (Li et al., 1992; Geiger
and Servaites, 1994). However, little is known about the activity of
starch biosynthetic enzymes or the expression levels of their genes
during the day/night cycle. Thus, the point at which
circadian regulation of starch synthesis might operate remains unclear.
In this study we cloned and characterized a gene encoding the GBSSI
protein. We show that in snapdragon (Antirrhinum majus) this
gene is present as a single copy and is responsible for GBSSI activity
in all organs and tissues in which amylose is synthesized. A detailed
study of the expression of this gene has been performed. Results show
high expression in meristematic tissues and precise spatial and
developmental regulation of its expression during flower development.
We also show a circadian regulation of GBSSI transcript
levels in leaves. GBSSI mRNA levels decrease at the end of
the day and cannot be detected during the night even when plants are
maintained under constant illumination. Sequence comparison of the
promoter region of the GBSSI gene with the promoters of other genes regulated by the circadian clock identify the presence of
conserved cis elements that have been shown to be critical components of this regulation. The integration of regulation of starch
synthesis into a more general circadian regulation of C metabolism is
discussed.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
Snapdragon (Antirrhinum majus stock JI:7, John Innes
Centre, Norwich, UK) was grown in a greenhouse under a natural
day/night regime. Plants cultured in growth cabinets were grown under a 16-h light/8-h dark photoregime at 23°C (day)/20°C (night), 70% RH, and a light intensity at the plant level of 120 µE
m 2 s1 supplied by white
fluorescent lamps.
Isolation of a cDNA Fragment Encoding GBSSI
A cDNA library made with mRNA from snapdragon leaves in  gt10
was screened with a cDNA fragment of the pea GBSSI gene (Dry et al., 1992). Filters were hybridized in 6× SSC, 0.1% SDS, 0.02% Ficoll, 0.02% PVP, and 5 µg/mL salmon sperm DNA at 55°C and washed in 2× SSC and 0.5% SDS at 55°C. From eight positive clones, the one
with the largest insert (2.3 kb) was selected, and the cDNA fragment
was cloned into the pBluescript II SK( ) plasmid.
Isolation of Genomic Clones
A snapdragon genomic library in EMBL4 was screened with the
2.3-kb cDNA fragment previously cloned. Filters were hybridized as
described for the cDNA library at 55°C and washed in 0.1× SSC and
1% SDS. Twelve positive clones were obtained and showed overlapping fragments by restriction analysis.
Sequencing
Sequences were determined according to the method of Sanger et al.
(1977) by double-stranded plasmid sequencing in pBluescript using the
T7-sequencing kit (Pharmacia Biotech).
Extraction and Gel Analysis of DNA and RNA
DNA was extracted from leaves of snapdragon according to the
method of Martin et al. (1985). Southern hybridizations were performed
as described by Sommer et al. (1985). Total RNA was extracted as
described previously by Prescott and Martin (1987) and was analyzed by
electrophoresis through formaldehyde gels and transferred to GeneScreen
Plus filters (NEN Life Science Products). Filter hybridizations were
performed according to the manufacturer's instructions.
RT-PCR Amplification
cDNA was amplified by PCR according to the method of Frohman et
al. (1989). First-strand cDNA was synthesized from 10 µg of total RNA
using the (dT)-17 adaptor as the primer. To stop the reaction the
sample was diluted to 1 mL with water. A 10-µL sample was taken for
amplification using oligonucleotides from different regions of the
GBSSI gene. Amplification involved 40 or 20 cycles with a
denaturation time of 1 min at 94°C, an annealing time of 90 s at
55°C, and an extension time of 3 min at 72°C.
In Situ Hybridization Analysis
In situ hybridization was performed as described by Bradley et al.
(1993). Digoxigenin labeling of RNA and detection of signal were
performed using Boehringer Mannheim methods and reagents (kit no.
1175-041) with some modifications as described previously by Coen et
al. (1990). Plasmids pSA301 and pSA302, containing 1 and 0.5 kb
encoding for amino acids 1 to 367 and 367 to 547, respectively, were
cut at the 5 end of the insert and transcribed in the presence of
digoxigenin-labeled nucleotides. The same plasmids cut at the 3 end
were used to transcribe the sense probes.
The resulting probes were hydrolyzed to generate fragments of about 150 bp, and sections of different tissues were probed. After the
hybridization signal was detected, sections were counterstained with Fluorescent Brightener 28 (Sigma) and mounted in Entellan mounting medium (Merck, Darmstadt, Germany). Sections were photographed through a Nikon Microphot-SA microscope using Kodak ASA 200 Ektachrome film.
Starch Measurement
Leaf starch was quantified as described previously by Lin et al.
(1988). Starch was converted to Glc by incubation with amyloglucosidase (from Aspergillus niger, Sigma), and Glc amount was
analyzed enzymatically using hexokinase and Glc-6-P dehydrogenase
(Jones et al., 1977).
Iodine Staining of Starch Granules
Tissue sections (7 µm) fixed and wax embedded, as described by
Bradley et al. (1993), were treated with xylene to remove the wax and
then stained with Lugol solution. Stained slides were rinsed
with distilled water, and starch granules were visualized using a zoom
stereomicroscope (model SZ4045TR, Olympus). Images were captured with a
color video camera (model JVC TK-C1381), using the MicroImage image
analysis software (Olympus).
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RESULTS |
Isolation of cDNA and Genomic Fragments Encoding for GBSSI
A cDNA fragment encoding for the GBSSI from pea (Dry et al., 1992)
was used to screen a gt10 cDNA library made from mRNA from
snapdragon leaves. Eight positive clones were isolated, and the clone
containing the largest cDNA fragment (2.3 kb) was selected for
sequencing. This fragment contains an open reading frame encoding a
608-amino acid polypeptide with a predicted molecular mass of 66,360 D. The deduced amino acid sequence is very similar to other GBSSI proteins
previously described: 84%, 75%, and 71% of amino acid identity to
GBSSI from potato, pea, and barley, respectively (Rohde et al., 1988;
Dry et al., 1992; Van der Steege et al., 1992), indicating that the
cDNA selected encodes a snapdragon GBSSI protein.
The 2.3-kb cDNA fragment was used to screen a EMBL4 snapdragon
genomic library, and 12 positive clones were isolated. Those clones
contained overlapping fragments as shown by restriction analysis (data
not shown). By Southern analysis a 5-kb EcoRI genomic fragment was selected and subcloned into pBluescript II SK(). This
fragment contained the GBSSI gene and part of its promoter region.
GBSSI Is Present in a Single Copy in Snapdragon
The possible presence of more than one gene encoding for GBSSI in
snapdragon was tested by Southern analysis. Genomic DNA was cut with
different restriction enzymes and hybridized with the 2.3-kb cDNA
fragment under low-stringency conditions (50°C hybridization
temperature and 2× SSC and 0.1% SDS washing buffer). Results obtained
are shown in Figure 1. Both
EcoRI and XbaI digestions gave single hybridizing
bands. BstXI and the double-digestion BstXI/XbaI rendered in both cases an intense band
and a very faint one. These less intense bands correspond to
hybridization with the 130 bp of the cDNA fragment used as a probe that
extends upstream of the BstXI site (see map of the region in
Fig. 1). These results indicate that the GBSSI gene is
present in a single copy in snapdragon and would be responsible for the
granule-bound starch synthase activity in all of the organs of the
plant.
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| Figure 1.
Restriction analysis of snapdragon genomic DNA.
Twenty micrograms of genomic DNA was cut with EcoRI,
BstXI, XbaI, and BstXI
plus XbaI and analyzed by Southern analysis using the
2.3-kb full-length GBSSI cDNA fragment as a probe. The
map at the bottom shows the cloned EcoRI genomic
fragment and the position for BstXI and
XbaI sites. The region covered by the cDNA probe is
indicated by a thick black bar.
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Expression Pattern of the GBSSI Gene
We first studied the expression of the GBSSI gene in
snapdragon by northern analysis. Figure
2A shows that the gene exhibits its
highest level of expression in leaves and is expressed to an equal
level in inflorescences (0.5 cm long), mature flowers, and fruits
7 d after pollination. Expression of the GBSSI gene in
these organs was corroborated by RT-PCR using an oligonucleotide from
the 3 end of the gene and another from the 3-untranslated region
(Fig. 2B). These results clearly indicate that GBSSI is expressed in all organs studied. Expression in roots was extremely low
and could be detected only by RT-PCR (Fig. 2B).
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| Figure 2.
Analysis of GBSSI expression in
different tissues. A, Northern analysis. Total RNA (30 µg) from
fruits (lane Fr), flowers (lane Fl), 0.5-cm-long inflorescences (lane
In), and leaves (lane L) was probed with the 2.3-kb
GBSSI cDNA fragment. Loading was controlled by reprobing
the filter with a cDNA fragment encoding the 18S rRNA from sunflower.
Transcript in root samples was undetectable and is not shown. B, RT-PCR
analysis of GBSSI transcript. Total RNA (10 µg) from
fruits (lane Fr), flowers (lane Fl), 0.5-cm-long inflorescences (lane
In), leaves (lane L), and roots (lane R) was used for single-strand
cDNA synthesis and subsequent amplification by PCR (40 cycles) using
oligonucleotides SA9 (955-936 bp upstream from the stop codon) and SA6
(65-45 bp downstream from the stop codon). PCR products were analyzed
by Southern analysis using the whole GBSSI cDNA fragment
as a probe. Loading in lane R was 10-fold higher than in the rest of
the lanes to compensate for the low amplification obtained in this
tissue.
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A more detailed study of the GBSSI expression pattern was
performed by in situ mRNA hybridization. We used two different cDNA fragments to obtain probes, one fragment of 1 kb comprising the region
encoding the first 367 amino acids and a 0.5-kb fragment containing the
sequence encoding the amino acids 367 to 547 of GBSSI. In both cases we
obtained the same pattern of expression in all of the tissues and
organs studied. These results are consistent with those shown above,
which show that GBSSI is encoded by a single-copy gene in
snapdragon.
Expression in leaves was located mainly in the palisade parenchyma
(Fig. 3A), being much lower or absent in
the spongy cells of the mesophyll and suggesting that transient starch
accumulation in leaves along the light period takes place almost
exclusively in the palisade cells. This idea was supported by iodine
staining of cross-sections of leaves, which showed accumulation of
starch granules in the palisade parenchyma but not in the spongy cells (data not shown). GBSSI was also expressed in the
parenchymatic tissue of the stem, located mainly in the subepidermal
layer of cells (Fig. 3B). It is interesting that the expression of
GBSSI was very intense in meristematic tissues, in both the
apical meristem and in floral primordia (Fig. 3, C and D). High
expression of GBSSI was detected in all three layers of the
apical meristem (Fig. 3E). Iodine staining of these meristematic
tissues showed starch accumulation in all regions where
GBSSI was expressed (data not shown). These results indicate
that meristematic tissues are able to synthesize their own reserve of
carbohydrate, which could contribute in a significant manner to
maintaining the elevated growth rates of these tissues.
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| Figure 3.
In situ hybridization of sections from different
tissues with the digoxigenin-labeled GBSSI RNA probe. A,
Transverse section of leaf. Arrow, Hybridization in the palisade
parenchyma. B, Longitudinal section of stem. GBSSI
expression in the subepidermal layer is indicated. C, Longitudinal
section of a 0.5-cm-long inflorescence. Hybridization in apical
meristem (am) and floral primordia (fp) is visible. D, Negative control
of C using the sense probe. E, Longitudinal section of apical meristem.
Expression in the three layers of the apical meristem (am), bracts, and
floral primordia (fp) is apparent. F, Floral primordium once the whorls
have been formed. Hybridization is visible in ovary (ovar), sepals
(sep), and petals (pet). G, Longitudinal section of a flower bud. A
clear expression in the four whorls, including stamen (stm) and anthers
(ant), is shown. H, Longitudinal section of a flower before anthesis.
GBSSI expression is restricted to the carpel. I
and J, Flower at the same developmental stage as in H. Dark-field exposure was used to show the absence of expression in
sepals (sep) and petals (pet) at this stage (J). I shows a negative
control using the sense probe. K, Magnification showing a cross-section
of an anther in a flower bud (same developmental stage as in G). L,
Cross-section of anther before anthesis (same stage as H-J). No signal
was detected at this developmental stage. M, Magnification of H showing
GBSSI expression in ovules (ovu). N, Magnification of a
longitudinal section of a capsule 7 d after pollination.
Expression in the seed endosperm is clearly visible.
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GBSSI was highly expressed in floral primordia before organ
differentiation. This high expression was maintained in subsequent developmental stages and was clearly visible in the four whorls in
flower buds (Fig. 3, F and G). However, prior to anthesis, expression
of GBSSI was lost in sepals, petals, and stamens and was
subsequently detected only in the carpel (Fig. 3, H-J). The expression
in the carpel was maintained throughout flower development and was also
visible in the capsule once the fruit was formed. At early
developmental stages there was clear expression of GBSSI in
anthers (Fig. 3K) that was completely abolished prior to anthesis (Fig.
3L). By contrast, expression in carpels and ovules was maintained throughout flower development (in Fig. 3, J, L, and M correspond to
carpel, anther, and ovule, respectively, at the same developmental stage), indicating different requirements for starch synthesis in these
two reproductive organs. In fruit GBSSI expression was also
detected in seeds (Fig. 3N), indicating that snapdragon accumulates starch as a reserve material in the seed endosperm (data confirmed by
iodine staining, data not shown).
The presence of starch in flowers before anthesis was determined by
iodine staining of flower sections at the same developmental stage as
those shown in Figure 3, H to J. Figure
4, A and C, shows the presence of starch
granules in the carpel and the style but also in sepals and petals.
These results could indicate that in sepals and petals there is a
modified form of starch granule lacking amylose. However, the
iodine-stained starch of sepals and petals did not show the red color
characteristic of amylose-free starch (Kuipers et al., 1994). Thus, the
starch in these organs at this stage might result from synthesis at
earlier developmental stages. In accordance with the in situ results,
starch granules were also detected in the carpel. It is worth noting
the high accumulation of starch in the funiculus, connecting the ovule
to the ovary wall, and in the nectary (Fig. 3B, arrows), a tissue where
carbohydrate metabolism is very active.
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| Figure 4.
Iodine staining of longitudinal section of flowers
before anthesis. Longitudinal sections of flowers at the same
developmental stage as in Figure 3, H to J. Iodine staining was used to
detect starch accumulation. A, Starch granule accumulation in the
carpel, as well as petals and sepals. B and C, Magnification of A
showing starch accumulation in carpel and petals, respectively.
Accumulation of starch granules in the funiculus (fun) and in the
nectary (nec) are indicated in B.
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Expression of GBSSI through the Day/Night Cycle
The main feature of starch synthesis in leaves is its fluctuating
character throughout the day/night cycle. It is well established that
starch is accumulated in chloroplasts during the light period and
mobilized in the dark, supplying C to the plant when the photosynthetic CO2 fixation is not operative. This behavior is
illustrated in Figure 5A. Fifteen-day-old
snapdragon plants were adapted to a 16-h light/8-h dark period for 1 month, and then two fully expanded leaves of the same node were
collected from successive plants at 2-h intervals throughout the cycle.
The content of starch was determined enzymatically as described in
``Materials and Methods''.
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| Figure 5.
Starch accumulation and GBSSI
expression in leaves during the day/night cycle. A, Starch accumulation
in leaves during a 16-h light/8-h dark photoperiod regime. The bar at
the bottom of the graph indicates the corresponding day/night periods:
day (open box), night (closed box), and period corresponding to night
but in which light was kept on (strippled box). B, Time course of
GBSSI expression during a 16-h light/8-h dark
photoregime. Arrows and numbers at the bottom of the bar
indicate the time when samples were collected for RNA isolation.
Loading was controlled by reprobing the filter with DNA encoding 18S
RNA from sunflower. Starch content and GBSSI transcript
levels in leaves were determined in three independent time-course
experiments and the same results were obtained.
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At the beginning of the light period, a 2- to 4-h lag interval was
observed before net accumulation of starch was detected (Fig. 5A).
During this phase there was a slight decrease in the level of starch
relative to the amount present at the end of the dark period. After the
initial lag, the rate of starch accumulation remained relatively steady
until approximately 2 h before the onset of the dark, when it
began to decline. The starch accumulation rate had also declined by the
end of the normal day even when the day period was extended (Fig. 5A,
second cycle). During such extended light periods, the accumulation of
starch stopped and starch levels remained steady with values
oscillating at approximately 0.42 mg starch/g fresh weight. These
results are in accordance with those reported by Li et al. (1992) for
sugar beet leaves and suggest an endogenous circadian regulation of
starch accumulation during the day/night cycle.
In the same time-course experiments, samples were collected for
isolation of total RNA, and GBSSI mRNA levels during the
cycle were analyzed by northern analysis. As shown in Figure 5B, the level of GBSSI mRNA was high at midday (Fig. 5B, lanes 2 and
6), was drastically reduced by the end of the day (Fig. 5B, lanes 3 and
7), and was almost undetectable during the night (Fig. 5B, lanes 1, 4, and 5). The GBSSI mRNA level was also severely reduced even
when the light period was extended to the second night (Fig. 5B, lanes
8 and 9). Finally, 4 h after the end of the time corresponding to
the second night period, the GBSSI transcript level was
restored (Fig. 5B, lane 10). These results point to an endogenous
circadian clock regulating GBSSI mRNA abundance, which, in
turn, match the pattern of starch accumulation in leaves during the
day/night cycle.
A light-entrained circadian control of GBSSI transcript
levels would not be expected to occur in an organ such as the root. To
test this hypothesis we compared the amounts of GBSSI mRNA in roots and leaves of plants collected at the midpoints in the day and
night. Figure 6 shows the result of an
RT-PCR (20 cycles of amplification) using RNA from leaves and roots.
Although the presence of GBSSI mRNA could not be detected in
leaves after 4 h of darkness (Fig. 5B), levels of GBSSI
transcripts in roots remained unaffected. These results indicate that
the mechanisms controlling GBSSI mRNA abundance in leaves
during the day/night cycle do not operate in roots.
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| Figure 6.
RT-PCR of GBSSI transcripts in
leaves and roots during day and night. Samples were collected at the
midpoint of the day (L) and night (D) periods. GBSSI
transcripts amplified by PCR (20 cycles) were analyzed by Southern
analysis. Loading in roots was 100 times greater than in leaves to
compensate for the different levels of expression of
GBSSI in these organs.
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DISCUSSION |
We have characterized the expression of the gene encoding GBSSI
(or waxy protein) in snapdragon. It is well established that this
enzyme is responsible for the synthesis of amylose in the starch
granule, and mutants defective in this activity show an amylose-free
starch (Kuipers et al., 1994; Denyer et al., 1995). A role for this
enzyme has also been demonstrated in the synthesis of amylopectin, and
mutants of Chlamydomonas reinhardtii lacking GBSSI activity
have a structurally modified amylopectin (Delrue et al., 1992). Studies
in several species have revealed the presence of distinct genes
encoding different isoforms in all of the enzymatic steps of starch
biosynthesis (Martin and Smith, 1995; Smith et al., 1997). In the case
of the GBSSI gene, Hirano and coworkers showed that the
waxy gene of rice is expressed in seeds and anthers exclusively (Hirano and Sano, 1991; Hirano et al., 1995), indicating that there are other isogenes responsible for the granule-bound starch
synthase activity in other parts of the plant. In pea two isoforms of
GBSSI have been detected that are differentially expressed in the plant
(Denyer et al., 1997).
Unlike the situation in pea and rice, we detected expression of the
GBSSI gene in all organs tested by northern blot
hybridization: leaves, inflorescences, flowers, and fruits (Fig. 2A).
RT-PCR analysis using an oligonucleotide of the 3 end of the gene and another of the 3-untranslated region confirmed the expression of
GBSSI in those tissues, as well as in roots (Fig. 2B).
Southern analysis of genomic DNA cut with different restriction enzymes clearly indicate that the GBSSI gene is present in a single
copy in snapdragon and is responsible for all GBSSI
transcripts.
Most of the studies of the expression of genes involved in starch
biosynthesis have been carried out in storage organs. In this case, the
levels of GBSSI mRNA peak later than other starch synthase
isoforms during pea embryo development (Dry et al., 1992), and genes
encoding for the distinct isoforms of the starch-branching enzyme are
differentially expressed during the development of pea embryo (Burton
et al., 1995) and wheat (Morell et al., 1997) and maize endosperm (Gao
et al., 1997). However, there is very little information about the
pattern of expression of the starch biosynthesis genes in tissues other
than storage organs. In this report we have shown that GBSSI
is highly expressed in the palisade parenchyma but is only weakly
expressed in the spongy cells. This result correlates with data
obtained by iodine staining of these sections, which showed that starch
granules accumulate mainly in the palisade cells (data not shown) and
indicate the dominant role of this tissue in the supply of C skeletons
derived from starch to the rest of the plant. GBSSI was
highly expressed and starch accumulated in meristematic tissues such as
apical meristems and floral primordia, suggesting that local
accumulation of starch could be necessary, in addition to the direct
supply of Suc from source organs, to maintain the high growth rate
characteristic of meristems.
GBSSI expression showed specific patterns during flower
development. Expression in petals and sepals was visible at early developmental stages (Fig. 3, F and G) but was absent before anthesis. However, accumulation of starch granules in sepals and petals at this
stage was detected by iodine staining (Fig. 4). Recently, plastid
ontogeny during petal development in Arabidopsis was described (Pyke
and Page, 1998). Young petals from unopened buds contain green
chloroplasts, but as the petal lamina develops and expands plastids
lose their chlorophyll and redifferentiate into leukoplasts. The loss
of GBSSI expression observed in snapdragon petals
before anthesis could also represent one aspect of the
redifferentiation of chloroplasts into leukoplasts. Our results
indicate that starch synthesized at earlier stages is accumulated in
plastids and may subsequently be used by these organs as a C and energy
source.
Expression of the GBSSI gene in carpel tissue was maintained
throughout flower development and differentiation of the capsule. The
actual accumulation of starch in this organ was confirmed by iodine
staining, which showed large accumulation of starch granules in the
funiculus, connecting the ovule to the ovary wall. Further studies of
the rate of starch synthesis and mobilization will be necessary to
determine the role of starch found in this organ in the process of
fruit formation. This could give new insights to understand some of the
alterations detected in starch-less mutants of Arabidopsis, in which a
low seed biomass accumulation was observed (Schulze et al., 1994).
Starch synthesis in leaves is tightly regulated through the so-called
C-partitioning process, which diverts newly fixed
CO2 to Suc in the cytoplasm or starch in the
chloroplast, depending on the levels of different metabolites that act
as allosteric regulators (for review, see Stitt and Quick, 1989).
Regarding the biosynthesis of leaf starch, it has been shown that this
allosteric control is exerted on the first enzyme, ADP-Glc
pyrophosphorylase. This enzyme is allosterically activated by
3-phosphoglycerate and inhibited by inorganic phosphate (for review,
see Preiss and Levi, 1980; Preiss and Sivak, 1996). This allosteric
control could account for the fluctuations of starch synthesis during
the diurnal period. Li et al. (1992) showed circadian regulation of
starch accumulation in sugar beet leaves. They observed that starch
synthesis stopped during the night even when plants were kept under
constant illumination, suggesting an endogenous circadian shift in C
allocation at the end of the day, which diverts newly assimilated C to
Suc synthesis and increases export at the expense of starch
accumulation. Similar results were obtained for the C translocation
during the day in the wild type and a starch-deficient mutant of
tobacco (Geiger et al., 1995). However, the specific mechanism
responsible for this response has not been identified.
Circadian regulation of other components of the C-partitioning process
has also been shown as modulation of Suc phosphate synthase activity by
a protein phosphatase (Jones and Ort, 1997). In this report we have
shown for the first time, to our knowledge, circadian regulation of the
mRNA abundance of a starch biosynthesis gene. Transcript levels of
GBSSI decreased at the end of the day and were almost
abolished during the night. This response was controlled by an
endogenous circadian clock, which could be observed when the light was
extended to the night period (Fig. 5B). This result suggests that the
preparation of the plant for the usual night period is more complex
than has been previously suggested, involving changes in C allocation
from chloroplasts to the cytosol, Suc synthesis, and expression of some
of the starch biosynthesis genes. Expression of GBSSI during
the day/night cycle was differentially regulated in distinct organs of
the plant. Thus, although expression in leaves was controlled by an
endogenous circadian clock, this mechanism did not operate in roots
(Fig. 6), reflecting the differences in C metabolism between aerial,
source organs such as leaves and dark, sink organs such as roots.
Synthesis of starch involves different enzymes, and it will be
interesting to establish whether there is coordinate regulation of the
other starch biosynthesis genes.
Sequence analysis has revealed the presence of cis elements
in the promoter region of snapdragon GBSSI (data not shown),
which have been previously shown to be involved in circadian control of
gene expression, such as the Circadian Clock Associated
1-binding site (Wang et al., 1997; Wang and Tobin, 1998), and
light regulation, such as the binding site for the GT-1 transcription
factor (Hiratsuka et al., 1994) or the G-box (Chattopadhyay et al.,
1998). Similar elements are also present in the promoter region of the
SBE2.1 gene of Arabidopsis encoding a type II
starch-branching enzyme and that has been recently shown to be light
regulated (Khoshnoodi et al., 1998). The presence of these elements in
the GBSSI promoter region suggests that fluctuations on the
GBSSI mRNA abundance observed in leaves could be mediated by
a circadian regulation of the gene transcription. However, a further
functional analysis of the GBSSI promoter will be necessary
to establish the role of those putative regulatory elements on the
circadian regulation of GBSSI expression, as well as a
possible light-mediated regulation of GBSSI expression.
| |
FOOTNOTES |
1
This research was supported by grant no.
PB95-1267 from the Dirección General de Enseñanza Superior,
by Junta de Andalucía group no. CVI-129, and by Spanish-British
Joint Action HB 1995-0042.
2
Present address: Instituto de Recursos Naturales
y Agrobiología. Avda Reina Mercedes no. 10, 41012-Sevilla,
Spain.
*
Corresponding author; e-mail jmromero{at}cica.es; fax
34-54-46-0065.
Received November 30, 1998;
accepted March 5, 1999.
| |
ABBREVIATIONS |
Abbreviations:
GBSSI, granule-bound starch synthase I.
RT, reverse-transcriptase.
| |
ACKNOWLEDGMENTS |
We wish to thank R. Carpenter and E. Coen for providing
snapdragon seeds and facilities for the in situ hybridizations and H. Sommer for providing the snapdragon genomic library. We are also
grateful to E. Coen, A. Smith, M. Sivak, and C. Martin for invaluable
critical reading and revision of the manuscript. We thank A. Herrero
and E. Flores for their encouragement and help. The greenhouse
facilities from the University of Sevilla (Spain) are also
acknowledged.
| |
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Abstract
Introduction