Plant Physiol. (1999) 119: 191-198
Metabolism of
D-Glycero-D-Manno-Heptitol,
Volemitol, in Polyanthus. Discovery of a Novel Ketose
Reductase1
Beat Häfliger,
Elsbeth Kindhauser, and
Felix Keller*
Institute of Plant Biology, University of Zurich, Zollikerstrasse
107, CH-8008 Zurich, Switzerland
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ABSTRACT |
Volemitol
(D-glycero-D-manno-heptitol,
-sedoheptitol) is an unusual seven-carbon sugar alcohol that
fulfills several important physiological functions in certain species
of the genus Primula. Using the horticultural hybrid
polyanthus (Primula × polyantha) as
our model plant, we found that volemitol is the major nonstructural carbohydrate in leaves of all stages of development, with
concentrations of up to 50 mg/g fresh weight in source leaves (about
25% of the dry weight), followed by sedoheptulose
(D-altro-2-heptulose, 36 mg/g fresh weight),
and sucrose (4 mg/g fresh weight). Volemitol was shown by the
ethylenediaminetetraacetate-exudation technique to be a prominent
phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of
the phloem sap carbohydrates, surpassed only by sucrose (63%).
Preliminary 14CO2 pulse-chase radiolabeling
experiments showed that volemitol was a major photosynthetic product,
preceded by the structurally related ketose sedoheptulose. Finally, we
present evidence for a novel NADPH-dependent ketose reductase,
tentatively called sedoheptulose reductase, in volemitol-containing
Primula species, and propose it as responsible for the
biosynthesis of volemitol in planta. Using enzyme extracts from
polyanthus leaves, we determined that sedoheptulose reductase has a pH
optimum between 7.0 and 8.0, a very high substrate specificity, and
displays saturable concentration dependence for both sedoheptulose
(apparent Km = 21 mM) and NADPH (apparent Km = 0.4 mM). Our
results suggest that volemitol is important in certain
Primula species as a photosynthetic product, phloem
translocate, and storage carbohydrate.
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INTRODUCTION |
Alditols (sugar alcohols or acyclic polyols) may be chemically
described as reduction products of aldose or ketose sugars. The most
prevalent plant alditols are the hexitols sorbitol, mannitol, and
galactitol. However, as many as 17 different alditols occur naturally
in higher plants (for review, see Bieleski, 1982
; Lewis, 1984; Loescher
and Everard, 1996). The lesser-known alditols are often restricted in
their occurrence but still fulfill important functions in those plants
where they do occur. Volemitol (Fig. 1)
is a good example of a less common but important alditol. This seven-carbon sugar alcohol seems to be confined to certain sections of
the genus Primula, so much so that it has been suggested as a useful chemotaxonomical marker (Kremer, 1978). Very little is known
about the physiology and metabolism of volemitol in primulas, except
that it was an early photosynthetic product in cowslip (Primula
veris) and oxslip (Primula elatior) (Kremer, 1978).
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| Figure 1.
Fischer projections of volemitol and its four
structurally related seven-carbon sugars. Nomenclature follows that of
Collins (1987); trivial names are underlined.
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The physiological roles of alditols are manifold and largely resemble
those of disaccharides and oligosaccharides. They include photosynthetic assimilation, translocation and storage of carbon, and
reducing power, as well as protection against different types of
stresses (for review, see Bieleski, 1982; Lewis, 1984; Loescher and
Everard, 1996; Stoop et al., 1996). The biosynthetic pathways of the
hexitols sorbitol (glucitol), mannitol, galactitol (dulcitol), and the
pentitol ribitol have been established in higher plants. They generally
use NADPH as a hydrogen donor and aldose phosphate as a hydrogen
acceptor, in concert with the corresponding phosphatases. One exception
might be galactitol, which was suggested to be formed directly from
unphosphorylated Gal (and NADPH) (Negm, 1986). Although all foliar
alditols are thought to be phloem-mobile (Lewis, 1984), this has only
been demonstrated for sorbitol, mannitol, and galactitol (Zimmermann
and Ziegler, 1975; Davis and Loescher, 1990; Moing et al., 1992; Flora
and Madore, 1993).
To expand our knowledge of alditol metabolism in higher plants beyond
that of hexitols, we studied the carbohydrate metabolism of polyanthus
(Primula × polyantha). This popular
horticultural hybrid of primrose (Primula
vulgaris), oxlip, and cowslip (Mabberley, 1997) was
chosen because preliminary experiments showed that its volemitol
content is very high, similar to that of the wild-type species, and
because it may be easily grown both outdoors and indoors.
We give a general overview on volemitol metabolism in polyanthus with
special emphasis on the role of volemitol in plant development and
phloem transport. We also report on a novel enzyme, a NADPH-dependent ketose reductase, which forms volemitol by the reduction
of sedoheptulose.
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MATERIALS AND METHODS |
Chemicals
Biochemicals were obtained from Sigma or Fluka unless stated
otherwise in the text. The following rare carbohydrates, which are not
commercially available, were generous gifts: volemitol from R. Honegger
(University of Zurich, Switzerland), P. Köll (University of
Oldenburg, Germany), S.J. Angyal (University of New South Wales,
Sydney, Australia), and T. Okuda (Okayama University, Japan);
sedoheptulose from P. Köll, coriose from T. Okuda, and D-glycero-D-manno-heptose
from S.J. Angyal. Sedoheptulose was also isolated from desalted cryo
sap of Sedum album leaves and coriose from Coriaria
japonica leaves harvested from the University of Zurich Botanical
Garden by HPLC purification on a Ca column (see below).
Plant Material and Growth
Polyanthus (Primula × polyantha Hort.)
plants were grown from seed (Kraft, Nesslau, Switzerland) in a mixture
of commercial standard soil and sand (10:1; v/v) in a greenhouse. After
about 40 d the plantlets had two to four leaves and were
transferred to plastic pots. The average day and night temperatures in
the greenhouse were 25°C ± 3°C and 12°C ± 2°C,
respectively, and the RH was 55% ± 5%/78% ± 6%
(day/night). In winter illumination was supplemented with incandescent
lamps (200 µmol m
2
s1). For some experiments, we used polyanthus
plants grown outdoors at the University of Zurich Botanical Garden.
Enzyme Extraction and Assays
Up to 1 g of freshly harvested plant material was finely
chopped with a razor blade and extracted on ice in a glass homogenizer containing 5 volumes of extraction buffer (20 mM Hepes/KOH,
pH 7.5, 5 mM DTT, 5 mM
MgCl2, 2% [w/v] PEG-20,000, and 2% [w/v]
PVP K30). The homogenate was centrifuged at 20,000g at 4°C
for 20 min. The supernatant was immediately centrifuge-desalted through Sephadex G-25 preequilibrated with the appropriate assay buffer (Helmerhorst and Stokes, 1980). This fraction was designated as desalted crude enzyme extract. In preliminary experiments the addition
of 0.1% (w/v) Triton X-100 in the extraction buffer and/or freezing
leaves in liquid N2 prior to extraction did not
have any demonstrable effect on the sedoheptulose reductase activity.
Sedoheptulose reductase activity was measured either
spectrophotometrically by monitoring the continuous oxidation of NADPH at 340 nm or chromatographically using a fixed-time assay based on the
quantification of the end product, volemitol, by HPLC-PAD. The
spectrophotometric assay mixture (1 mL) contained 25 mM
sedoheptulose, 1 mM NADPH, extraction buffer, and 500 µL
of desalted crude enzyme extract. The chromatographic assay mixture
(100 µL) was composed similarly but contained only 50 µL of enzyme
extract. Boiling in a water bath for 5 min stopped the assay. After
centrifugation at 14,000g at 4°C for 3 min, the reaction
mixture was desalted and volemitol was determined by HPLC-PAD as
described below. One unit of sedoheptulose reductase activity
corresponds to the amount of enzyme that catalyzed the formation of 1 µmol of product (NADP or volemitol) per minute under the assay
conditions chosen.
Carbohydrate Extraction and Analysis
Water-soluble carbohydrates were extracted by the cryo-sap method
and immediately desalted and liberated from phenols by the mixed-bed
microfiltration method described previously (Bachmann et al., 1994).
Carbohydrates were routinely determined by HPLC-PAD using
cation-moderated partitioning chromatography on a Ca-carbohydrate column (300 × 7.8 mm; model SS-100, Benson Polymeric, Reno, NV) with 50 mg L1 Ca-EDTA as the eluant (at 0.6 mL
min1 and 90°C). For additional identification
of the peaks obtained by cation chromatography, anion-exchange
chromatography using a CarboPak MA1 column (250 × 4 mm) fitted
with a CarboPak MA1 guard column (50 × 4 mm), both from Dionex
(Sunnyvale, CA), with 0.6 M NaOH as the eluant (at 0.4 mL
min1, at room temperature) and PAD was used.
The HPLC systems used have been described in detail previously (Keller
and Ludlow, 1993; Bachmann et al., 1994). Quantification of the
water-soluble carbohydrates was by the external standard method using
authentic standards. Because sedoheptulose cannot be obtained in
crystalline form, it was quantified using the response factor of
mannoheptulose. For the study on the ontogenetic pattern of
nonstructural leaf carbohydrates (Fig. 3), we used lyophilized leaf
tissue and followed exactly the procedure described for pigeon pea
(Keller and Ludlow, 1993).
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| Figure 3.
Changes of fresh weight and nonstructural
carbohydrate concentrations in polyanthus leaves during development.
The plant analyzed was greenhouse-grown (25°C/15°C; day/night), 4 months old (postflowering), and had reached its 12-leaf stage. The
leaves were harvested about 3 h into the light period. The
carbohydrates were determined by the HPLC-PAD method described in
``Materials and Methods''. Two successive leaves were combined for
analysis. A, Leaf fresh weight (fwt) ( ) and starch ( ). B,
Volemitol ( ), sedoheptulose ( ), Suc ( ), Glc ( ), and Fru
( ).
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Phloem Exudation
Phloem exudates were obtained by the EDTA method described
previously (Bachmann et al., 1994). The three youngest fully expanded leaves of 3-month-old plants were excised and their petioles were recut
under a collection solution of 5 mM phosphate buffer and 5 mM EDTA, pH 7.5. The petioles were quickly placed into
small plastic dishes (3.2 × 2.6 × 1.6 cm) containing 5 mL
of fresh collection solution per leaf. About 0.6 mm of the petiole was
immersed in the solution. The samples were kept in an airtight,
translucent plexiglass (light incubation) or black, plastic (dark
incubation) chamber lined with moist filter paper to maintain high
humidity. At the times indicated (usually in 3-h intervals), 4.5 mL of
the exudation solution was withdrawn and immediately replaced by the same volume of new collection solution. Exudation solutions were desalted and analyzed by HPLC-PAD as described above. The amounts of
exuded carbohydrates are expressed as micromoles per gram fresh weight
of the leaves at the beginning of the experiment.
In preliminary experiments the optimum composition of the collection
solution was determined; EDTA concentration was varied between 0 and 20 mM, and pH was varied between 6.0 and 8.0. Virtually no
carbohydrates exuded when tap water was used as the collection solution. When the EDTA-containing collection solution was replaced with tap water after 4 h of EDTA exudation, exudation slowed and came to a complete halt within the next 19 h.
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RESULTS |
Volemitol Occurrence
Water-soluble carbohydrates were analyzed by HPLC-PAD (Fig.
2, A and B). Table
I shows that the two main soluble
carbohydrates detected in leaves of polyanthus, oxslip, and
Primula juliae (all members of the section
Primula) were volemitol and sedoheptulose; Suc, Glc, and Fru
were also present but in much smaller concentrations. In leaves of
Primula denticulata (section Denticulata), Suc,
Glc, and Fru but not volemitol (or any other known alditol) and only traces of sedoheptulose were found. In a small survey, the presence of
volemitol was further confirmed for leaves of an additional two members
of the section Primula, cowslip and primrose; its absence
was observed in leaves of 13 Primula species belonging to
the sections Auriculastrum, Aleuritia,
Proliferae, Sikkimensis, Oreophlomis,
and Muscarioides (data not shown). Our results on volemitol
occurrence are identical to those published by Kremer (1978), and
confirm the importance of volemitol as a chemotaxonomic marker of the
genus Primula.
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| Figure 2.
HPLC chromatograms of a carbohydrate standard
(A), water-soluble carbohydrates (B), and sedoheptulose reductase
reaction products (C) of fully mature polyanthus leaves. Carbohydrates
were extracted by the cryo-sap method and determined by HPLC-PAD. The
sedoheptulose reductase assay was performed as described in
``Materials and Methods''. The control in C was a boiled enzyme blank
incubated for 30 min. 1, Raffinose; 2, Suc; 3, galactinol; 4, Glc; 5, sedoheptulose; 6, Fru; 7, myo-inositol; 8, volemitol; 9, perseitol.
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Table I.
Main soluble carbohydrates and sedoheptulose
reductase activities of leaves of some Primula species
Youngest fully mature leaves were harvested in March from plants grown
under natural conditions in the University of Zurich Botanical Garden.
Data are means ± SE from three plants.
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Leaf volemitol concentrations of plants harvested in spring were high,
ranging from 23 to 50 mg g1 fresh weight
(13%-25% of the dry weight) (Table I). Sedoheptulose concentrations
were also quite high, but generally a bit lower than volemitol
concentrations, ranging from 14 to 36 mg g1
fresh weight (7%-18% of the dry weight). The volemitol
concentrations of mature leaves of wild-grown polyanthus plants were
about twice as high in March (flowering) as in June (postflowering),
indicating possible seasonal variations (data not shown); and volemitol
concentrations of leaves of warm-grown greenhouse plants were generally
lower (by a factor of 2-3) than those of cold-grown greenhouse or
wild-grown spring plants, also indicative of seasonal variation (Fig.
3; F. Keller, unpublished
observations).
Changes of Leaf Nonstructural Carbohydrate Concentrations during
Development
To study the ontogenetic pattern of leaf carbohydrate
concentration, we analyzed a 4-month-old greenhouse-grown
(25°C/12°C day/night temperatures) plant that had reached its
12-leaf stage (postflowering). The youngest leaves analyzed (leaves 1 and 2) were in the center of the rosette, still partly unfolded and
pale green. Leaves 7 and 8 were fully expanded and totally green,
whereas the outer leaves (11 and 12) were slightly smaller and starting to senesce. Fig. 3A shows the gradual increase of the leaf biomass with
age, which reached a maximum in leaves 9 and 10.
Volemitol was the dominating carbohydrate in leaves at all stages of
development, gradually increasing from 2.3 mg
g1 fresh weight in the youngest leaves (1 and
2) to 15.4 mg g1 fresh weight in the oldest
leaves (11 and 12), and contributing between 43% and 73% to the
totality of the nonstructural carbohydrates (Fig. 3B). The second most
prominent soluble carbohydrate was sedoheptulose. Unlike volemitol, its
concentration remained constant with development (between 1.1 and 2.5 mg g1 fresh weight). The common sugars Suc,
Glc, and Fru were minor components. Starch was also a minor
carbohydrate in young to mature leaves but became prominent in the
oldest two leaves (12.3 mg g1 fresh weight;
Fig. 3A).
Similar patterns of nonstructural carbohydrate distributions within
polyanthus rosettes were observed in plants of a variety of ages (from
the 2-leaf to the 15-leaf stage) and growing conditions (cold/warm).
Volemitol concentrations were always low in sink leaves and high in
source leaves, whereas no distinct concentration gradients were
apparent for sedoheptulose. The absolute values of the carbohydrate
concentrations, however, varied greatly with the history of the plants
(F. Keller, unpublished results).
Phloem Mobility of Volemitol
The diurnal course of carbohydrate exudation from mature
polyanthus leaves is shown in Figure 4.
The two main phloem-mobile carbohydrates were Suc and volemitol. No
clear diurnal fluctuations in the levels of these two carbohydrates
were seen, with the possible exception of a transient increase in the
Suc level during the first hours of the light period. Minor components
found in the phloem exudates were Fru and Glc, with traces of
sedoheptulose also seen. Because the two hexoses were present in about
equal molar amounts and they are generally not phloem mobile, we
attribute their origin to invertase products of translocated Suc rather than the phloem. Comparing average exudation rates calculated from the
data shown in Figure 4, of the 106 nmol (31.5 µg) of carbohydrates
that were exuded per gram of leaf fresh weight per hour, about 58%
were found in Suc and 21% in volemitol.
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| Figure 4.
Diurnal pattern of soluble carbohydrates found in
phloem exudates of mature leaves of 3-month-old polyanthus plants.
Exudation was performed with excised leaves placed into a 5 mM EDTA solution at pH 7.5. The carbohydrates were
determined by the HPLC-PAD method described in ``Materials and Methods''. Black bar indicates nighttime. fwt, Fresh weight.
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In a similar experiment in which the phloem exudates were collected
over a 6-h period, the average exudation rate was 143 nmol (41.5 µg)
carbohydrate g1 fresh weight, and Suc and
volemitol made up 67% and 27%, respectively, of the total
carbohydrates exuded (also calculated on a molar basis). The
carbohydrate composition of the phloem is thus clearly distinct from
that of the whole-leaf tissue, where volemitol was the main component
(71% of the total soluble leaf carbohydrate on a molar basis),
followed by sedoheptulose (14%), and Suc was a minor component (2%)
(data not shown, see Table I).
Sedoheptulose Reductase, a Novel Enzyme
HPLC revealed that when a desalted crude enzyme extract of mature
polyanthus leaves was incubated in the presence of sedoheptulose and
NADPH, volemitol was formed (Fig. 2C). Under the assay conditions chosen, the reaction was linear for at least 30 min, and doubling the
amount of enzyme also doubled the sedoheptulose reductase activity
(data not shown). Similar reaction rates were obtained when the
oxidation of NADPH to NADP was monitored spectrophotometrically at 340 nm. When the pH of the reaction mixture was varied between 4.0 and 9.0 using the four buffer systems Mes-KOH (pH 5.5-6.5), Hepes-KOH (pH
6.5-7.5), Tricine-KOH (pH 7.5-9.0), and McIlvaine (pH 4.0-7.0), a
broad pH optimum between 7.0 and 8.0 was determined (data not shown).
The temperature dependence of sedoheptulose reductase showed a maximum
at 45°C (data not shown). Sedoheptulose reductase activity decreased
slowly with decreasing temperature and retained 44% of its maximum
activity, even at 14°C. Sedoheptulose reductase showed saturable
concentration dependence for both the substrate, sedoheptulose (Fig.
5A) with an apparent
Km value of 20.8 mM,
and the co-substrate, NADPH (Fig. 5B) with an apparent Km value of 0.395 mM.
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| Figure 5.
Effect of sedoheptulose (A) and NADPH (B)
concentration on polyanthus leaf sedoheptulose reductase activity.
Enzyme activities were determined by the HPLC-PAD method described in
``Materials and Methods''. The concentrations of NADPH and
sedoheptulose were 1 and 25 mM in A and B, respectively.
Insets show the Hanes-Woolf replots of the data. fwt, Fresh weight.
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The specificity of sedoheptulose reductase was very high for both the
cosubstrate and the substrate. Enzymatic sedoheptulose reduction was
observed only with NADPH, not with NADH (at 1 mM) as the
reductant (data not shown). Of the 14 different substrates tested, only
2 showed sedoheptulose reductase activity, sedoheptulose and
D-xylulose. The survey was performed in triplicate under
standard assay conditions, with different polyanthus enzyme samples
containing sedoheptulose reductase activities between 30 and 100 milliunits g1 fresh weight (as measured with
sedoheptulose). No products were detected by the sensitive HPLC-PAD
assay from the seven-carbon sugars,
D-glycero-D-manno-heptose,
mannoheptulose (D-manno-2-heptulose), coriose (D-altro-3-heptulose) (all
potential precursors structurally related to volemitol [Fig. 1]),
D-mannoheptose
(D-glycero-D-galacto-heptose), and D-glucoheptose
(D-glycero-D-gulo-heptose),
the seven-carbon sugar phosphate,
D-sedoheptulose-7-phosphate, and the six-carbon sugars, D-Glc, D-Fru,
L-sorbose, D-psicose,
D-tagatose, the pentulose D-ribulose, as well as the ubiquitous
disaccharide, Suc. D-Mannoheptose, mannoheptulose, coriose, Glc, and Fru were also tested with the spectrophotometric assay for their ability to oxidize NADPH, and did
not show any detectable activity. The sedoheptulose reductase activity
with the pentulose D-xylulose was between 7% and
10% of the rate with sedoheptulose and was observed with both the chromatographic and spectrophotometric assay. On the Ca column, the
product coeluted with arabinitol, which is one of the two expected
xylulose reduction products (the other is xylitol).
To further test whether sedoheptulose is the precursor of volemitol
synthesis in planta, preliminary photosynthetic
14CO2 pulse-chase,
radiolabeling experiments were performed with polyanthus leaf discs.
After a 4-min pulse, most of the label of the neutral soluble fraction
was in sedoheptulose, followed by Glc, Suc, and Fru; volemitol was
still unlabeled. Some label started to appear in volemitol after a
10-min pulse. A more comprehensive study of the kinetics of
sedoheptulose and volemitol production is under way.
Volemitol does not seem to be exclusively synthesized in green tissue.
When different parts of 6-month-old polyanthus plants were analyzed for
sedoheptulose reductase activity, leaf laminae, fleshy nongreen
petioles, and roots all displayed comparable sedoheptulose reductase
activities in the range of 50 to 100 milliunits
g1 fresh weight (data not shown).
Sedoheptulose reductase activity was only found in volemitol-containing
Primula species (Table I). Leaf enzyme extracts of P. denticulata, which lack volemitol, did not show any measurable activity. Recombination experiments mixing extracts of leaves of
polyanthus and P. denticulata gave no indication of
inhibitors. Enzyme extracts of perseitol-containing and
volemitol-lacking avocado leaves also did not show any sedoheptulose
reductase activity (data not shown), supporting the conclusion that
sedoheptulose reductase is typical for volemitol-containing tissue.
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DISCUSSION |
At the onset of our study, quantitative data on the occurrence of
soluble carbohydrates in Primula species were scarce and based on somewhat outdated methods. At best, they had been obtained using one- or two-dimensional paper chromatography or TLC (e.g. Kremer,
1978; Beck and Hopf, 1990). In an attempt to upgrade to HPLC as the
main analytical tool, we compared the performance of two HPLC columns
to separate the carbohydrates expected to be present in primulas: a
Ca-loaded ion-moderated partition column and an anion-exchange column
at high pH. The Ca column proved to be suitable, reliable, and rapid
for routine work (Fig. 1). It must be emphasized, however, that this
column was not totally selective; for example, it was unable to
separate sedoheptulose from Gal. Fortunately, by using the more
selective (but slower and more delicate) MA1 anion-exchange column, we
found that Gal is present only in trace amounts (more than 2 orders of
magnitude lower than sedoheptulose) in Primula leaves. The
identity of volemitol and sedoheptulose isolated from polyanthus leaves
was confirmed by GC-MS (performed by Dr. A. Richter of the University
of Vienna) and that of volemitol by 1H-NMR
spectroscopy (performed by Dr. P. Rüedi of the University of
Zurich).
The volemitol concentration in Primula leaves (Table I) was
similar to that of many other alditol plants (up to 25% of the dry
weight; Dietz and Keller, 1997). Such a high concentration is clear
evidence for the role of volemitol as a storage carbohydrate. As with
other alditols, this implies that volemitol ideally combines storage of
carbon and reducing power, because alditols are more reduced than
sugars.
Of special interest was the occurrence of high concentrations of free
sedoheptulose (up to 18% of the dry weight, Table I) and the
invariable co-occurrence of sedoheptulose and volemitol in
Primula leaves. Although sedoheptulose is universal in the plant kingdom, it is normally only found in the form of its mono- and
bisphosphate esters, in the pentose phosphate cycles. Free sedoheptulose has rarely been described and seems to be confined to
selected vascular plants, mostly of the Crassulaceae (Hegnauer, 1964; Okuda and Mori, 1974). Only speculations exist about the physiological roles of sedoheptulose; they include carbon storage and
cryoprotection (Kull, 1967). In this paper we present evidence for a third role of sedoheptulose: as an important metabolic precursor. Therefore, the coexistence of volemitol and sedoheptulose in primula is
not surprising.
The occurrence of volemitol in phloem exudates of polyanthus leaves is
a new discovery. Although expected because hexitols such as mannitol
(Davis and Loescher, 1990; Flora and Madore, 1993; Flora and Madore,
1996) and sorbitol (Moing et al., 1992, 1997) have been shown to be
important phloem-mobile carbohydrates, no heptitols have actually been
demonstrated to play the same role.
The molar Suc-to-volemitol ratio in the phloem sap of polyanthus was
about 2.5, which is similar to the Suc-to-mannitol ratio in parsley
phloem (Flora and Madore, 1996), but higher than the Suc-to-mannitol
ratios reported for celery phloem (about 0.5; Daie, 1987). In the woody
Rosaceae species peach, the molar Suc-to-alditol ratio in the phloem is
even more in favor of the alditol sorbitol with values up to 0.25 (Moing et al., 1997). The implications of the high phloem
Suc-to-volemitol ratio are not totally clear, but may be an indication
that in polyanthus, the primary role of volemitol is storage and that
of Suc is translocation.
A further physiological role of volemitol might be cryoprotection. In
temperate regions, wild-growing polyanthus is found mainly in open
grassy habitats, where it flowers in early spring (March/April),
undergoes a phase of leaf expansion in early summer (June/July), and
overwinters with young green leaves. The observed winterhardiness
implies freezing tolerance of both the aboveground and the underground
organs. Volemitol is a possible candidate as a cryoprotectant because
it is present in both types of organs at considerable concentrations,
and because nonreducing carbohydrates such as Suc, raffinose
oligosaccharides, and various polyols have been suggested to be
involved in the acquisition of freezing tolerance (for review, see Popp
and Smirnoff, 1995; Keller and Pharr, 1996; Loescher and Everard,
1996). However, because no data in support of the role of volemitol as
a cryoprotectant are available, it can only be considered an
interesting possibility.
The finding that volemitol is synthesized by the action of a
NADPH-dependent ketose reductase is somewhat surprising because the
best-studied reductases involved in vascular plant alditol biosynthesis
use aldoses (not ketoses) and phosphate esters (not free sugars) as
their substrates; i.e. Glc-6-P is used for sorbitol formation, Man-6-P
for mannitol formation, and Rib-5-P for ribitol formation (for review,
see Loescher and Everard, 1996). Exceptions have been described;
galactitol is thought to be synthesized by a NADPH-dependent aldose
reductase (albeit with an unusually high Km
value of 227 mM for Gal; Negm, 1986) and an
alternative pathway for sorbitol synthesis has been proposed, which
involves a free ketose (Fru) as the substrate and NADH as the
cosubstrate (Doehlert, 1987). The discovery of sedoheptulose reductase
described here, which involves a free ketose as the substrate, is an
exciting expansion of our knowledge of plant alditol biosynthesis.
Whether such a nonsugar-phosphate/ketose biosynthetic pathway is
typical for plant heptitols (e.g. perseitol synthesis from
mannoheptulose) is an intriguing question that will have to be answered
in the future.
The involvement of sugar phosphates in volemitol biosynthesis cannot be
totally ruled out, because only one potential candidate, sedoheptulose-7-phosphate, was tested and it did not show any activity
with either NADH or NADPH as cosubstrates. Other seven-carbon sugar
phosphates such as mannoheptulose-phosphate, coriose-phosphate, and
D-glycero-D-manno-heptose-phosphate
are theoretically possible (Fig. 1), but could not be tested because
they are not commercially available. It is noteworthy that in the
benthic marine brown alga Pelvetia canaliculata, volemitol
was shown to be synthesized from a sugar phosphate,
sedoheptulose-7-phosphate, by the action of a NADH-dependent reductase
via volemitol-1-phosphate (Kremer, 1977). Whether this type of
reductase is limited to certain brown algae is not known. It would be
interesting to do a comparative study of the volemitol biosynthetic
pathways expected to occur in the variety of known volemitol-containing
organisms ranging from basidiomycetes and yeasts to algae, lichens,
liverworts, and vascular plants (for review, see Bieleski, 1982; Lewis,
1984).
In conclusion, we have shown that: (a) volemitol and sedoheptulose are
the two main nonstructural carbohydrates, (b) volemitol and
sedoheptulose are photosynthetic products, (c) volemitol is phloem-mobile (like Suc), and (d) volemitol biosynthesis proceeds by a
novel, NADPH-dependent, ketose reductase, tentatively called sedoheptulose reductase because of its high substrate specificity. The
stage is now set to further study and define the physiological roles of
volemitol and the subcellular compartmentation of its biosynthetic
pathway, as well as to characterize sedoheptulose reductase in depth at
both the biochemical and molecular levels.
| |
FOOTNOTES |
*
Corresponding author; e-mail fkel{at}botinst.unizh.ch; fax
41-1-634-8204.
Received July 13, 1998;
accepted October 2, 1998.
1
This work was supported by the Swiss National
Foundation.
| |
ABBREVIATIONS |
Abbreviations:
PAD, pulsed amperometric detection.
sedoheptulose, D-altro-2-heptulose.
volemitol, D-glycero-D-manno-heptitol,
-sedoheptitol.
| |
ACKNOWLEDGMENTS |
We thank Mason Pharr for critical comments on the manuscript and
Helen Greutert for expert technical assistance.
| |
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