Plant Physiol. (1998) 116: 709–714
Phloem Transport of Fructans in the Crassulacean Acid
Metabolism Species Agave deserti1
Ning Wang2 and Park S. Nobel*
Department of Biology, University of California, Los Angeles, California 90095–1606
such species. Because the Suc concentration in the photosynthetic cells is often lower than or similar to that in the
phloem for these species, symplastic phloem loading of Suc
via plasmodesmata would also not occur.
To resolve this dilemma of symplastic phloem loading,
Turgeon (1991) has proposed a polymer-trapping model
for loading of Suc and RFO, in which Suc in the photosynthetic cells diffuses via plasmodesmata down its concentration gradient to the bundle-sheath cells and then to
intermediary cells (specialized companion cells), where raffinose and stachyose are synthesized. Raffinose and stachyose then diffuse from the intermediary cells to sieve tubes
down their concentration gradients but cannot diffuse back
into bundle-sheath cells because the channel size of plasmodesmata between intermediary cells and bundle-sheath
cells is too small for their passage. This polymer-trapping
model is supported by ultrastructural studies, the concentration of RFO in the intermediary cells, immunolocalization of enzymes responsible for RFO synthesis, and other
physiological evidence (van Bel, 1993; Haritatos and Turgeon, 1996). However, it is not clear whether such a model
can also apply to species that transport oligosaccharides
other than RFO (such as fructans) or to monocotyledonous
species.
Fructans are soluble polymers of Fru with a terminal Glc
residue. They function as the main storage carbohydrates
in 15% of flowering plant species, including many economically important crops (Pollock and Cairns, 1991; PilonSmits et al., 1996; Wiemken et al., 1996). Fructans also play
roles in osmoregulation during drought (Spollen and Nelson, 1994; Wiemken et al., 1996) and can act as protectants
against dehydration imposed by drought or freezing (Wiemken et al., 1996). Despite the important functions and wide
distribution of oligofructans in flowering plants, virtually
no information is available concerning oligofructan transport in higher plants.
Fructans are synthesized and stored in the stems of
agaves (Aspinall and Gupta, 1959; Dorland et al., 1977;
Bhatia and Nandra, 1979). The main function of fructans in
the stems of such CAM plants is storage, as for C3 and C4
plants, and they may also act as osmoprotectants during
drought. However, it is unknown whether fructans occur
in the phloem of agaves. When we studied source-sink
photosynthate partitioning for the CAM species Agave deserti, preliminary observations indicated that oligofructans
Four oligofructans (neokestose, 1-kestose, nystose, and an unidentified pentofructan) occurred in the vascular tissues and
phloem sap of mature leaves of Agave deserti. Fructosyltransferases
(responsible for fructan biosynthesis) also occurred in the vascular
tissues. In contrast, oligofructans and fructosyltransferases were
virtually absent from the chlorenchyma, suggesting that fructan
biosynthesis was restricted to the vascular tissues. On a molar basis,
these oligofructans accounted for 46% of the total soluble sugars in
the vascular tissues (sucrose [Suc] for 26%) and for 19% in the
phloem sap (fructose for 24% and Suc for 53%). The Suc concentration was 1.8 times higher in the cytosol of the chlorenchyma cells
than in the phloem sap; the nystose concentration was 4.9 times
higher and that of pentofructan was 3.2 times higher in the vascular
tissues than in the phloem sap. To our knowledge, these results
provide the first evidence that oligofructans are synthesized and
transported in the phloem of higher plants. The polymer-trapping
mechanism proposed for dicotyledonous C3 species may also be
valid for oligofructan transport in monocotyledonous species, such
as A. deserti, which may use a symplastic pathway for phloem
loading of photosynthates in its mature leaves.
Plant growth depends on the supply of photosynthates
via the phloem to sink organs. The process of photosynthate delivery from photosynthetic cells to the phloem of
source organs (phloem loading) is an important determinant of such growth, and numerous studies have been
conducted to understand its mechanism (Giaquinta, 1983;
van Bel, 1993). In species for which Suc is the only transported sugar, phloem loading may occur by co-transport of
Suc and protons from the apoplast (i.e. apoplastic loading;
Giaquinta, 1983; van Bel, 1993; Grusak et al., 1996). For
other species, however, RFO and other carbohydrates in
addition to Suc are transported in the phloem (Turgeon et
al., 1975; Ziegler, 1975; Fisher, 1986; Flora and Madore,
1993). In many of these species, such as Coleus blumei,
Cucurbita pepo, and Olea europaea, phloem loading is not
sensitive to the inhibitor of active Suc transport,
p-chloromercuriphenylsulfonic acid; therefore, such a
mechanism of apoplastic phloem loading may not apply to
1
This research was supported by the Office of Health and
Environmental Research, U.S. Department of Energy, Program for
Ecosystem Research (grant no. DE-FG03-93ER61686).
2
Present address: DuPont Central Research and Development,
Experimental Station, P.O. Box 80328, Wilmington, DE 19880 –
0328.
* Corresponding author; e-mail [email protected]; fax
1–310 – 825–9433.
Abbreviations: DP, degree of polymerization; RFO, raffinosefamily oligosaccharides.
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Wang and Nobel
occur in the phloem sap and vascular tissues of mature
leaves. Such findings raise important questions about where
these oligofructans are synthesized and how they are loaded
into the phloem of mature leaves. For example, is the
phloem loading of photosynthate apoplastic or symplastic?
The present study was initiated to determine the cellular
site(s) for oligofructan biosynthesis and distribution and to
test whether the polymer-trapping model (Turgeon, 1991)
can also explain phloem loading of oligofructans in mature
source leaves of the monocotyledon A. deserti. Thus, the
distribution of mono- and oligosaccharides among different tissues of mature source leaves of A. deserti was examined to see whether oligofructans are specifically located in
the phloem. The activities of enzymes responsible for fructan biosynthesis and hydrolysis were analyzed to determine the site(s) of fructan biosynthesis. The concentration
gradients of mono- and oligosaccharides along the phloemloading pathways from photosynthetic cells to the sieve
tubes/companion cells were estimated to establish a possible loading mechanism for photosynthates in mature
leaves of A. deserti.
MATERIALS AND METHODS
Twenty plants of Agave deserti Engelm. (Agavaceae) with
10 to 12 unfolded leaves averaging 28 cm in length were
maintained in 14-L pots in a greenhouse at the University
of California, Los Angeles. The mean total daily PPFD was
38 mol m22 d21, corresponding to a mean instantaneous
value of 800 mmol m22 s21, and the daily maximum/
minimum air temperatures averaged 28/16°C, respectively
(North and Nobel, 1995). The plants were watered twice
weekly with 0.1-strength Hoagland solution. Two months
before the experiments, the plants were transferred to Conviron E-15 environmental growth chambers (Controlled
Environments, Pembina, ND) with daily maximum/minimum air temperatures of 25/15°C, respectively. The photoperiod was 12 h, with a total daily PPFD of about 35 mol
m22 d21. The plants were again watered twice weekly with
0.1-strength Hoagland solution. Such conditions are near
the optimum for the growth of A. deserti (Nobel, 1988).
Tissue Harvest and Phloem Sap Collection
Mature leaves of A. deserti have chlorenchyma layers
about 1 mm thick on both the upper and lower surfaces,
with 1 to 2 mm of water-storage parenchyma in between,
which allows for ready separation and collection of these
two tissues. To assess diurnal changes of carbohydrates
and malate in the chlorenchyma and in the water-storage
parenchyma, 0.1 to 0.5 g of each tissue was harvested
individually at various times of the day from the middle of
mature source leaves of A. deserti using a razor blade.
One-half of the harvested chlorenchyma or water-storage
parenchyma was immediately frozen using dry ice and
stored at 270°C, and the other half was dried at 80°C for
48 h to determine water content.
To obtain the vascular tissues, which in mature leaves
occur as longitudinal veins extending from the leaf base to
the tip without direct lateral vascular connections, sections
Plant Physiol. Vol. 116, 1998
with a width of about 1 mm were cut transversely across
the middle of mature leaves. These sections, which contained the chlorenchyma, water-storage parenchyma, and
vascular tissues, were frozen on dry ice and then dehydrated in a freeze-drying system (Labconco, Kansas City,
MO) prior to storage at 270°C. A syringe needle of about
0.35 mm in diameter was inserted into the vascular strand
area, which is readily visible under a stereomicroscope.
Cores of vascular tissues, which were contaminated with a
small amount of chlorenchyma and water-storage parenchyma, were collected by repeating such insertions and
were weighed with a microbalance (ATI Cahn, Boston,
MA). The dissected vascular tissues were stored at 270°C
for measurements of metabolites and enzyme activities.
Phloem sap was collected with severed stylets of the
scale insect Ovaticoccus californicus McKenzie, which naturally infests mature leaves of A. deserti, using a method
similar to that developed for Opuntia ficus-indica (Wang
and Nobel, 1995). Colonies of the insects that infested the
middle region of mature leaves were gently wiped away
with tissue paper soaked in 80% ethanol, leaving the severed stylets protruding from the leaf surface, and mineraloil-filled wells were constructed covering the areas containing the severed stylets. Phloem sap was collected once
every 2 to 3 h to minimize microorganism contamination.
In many cases, phloem sap exuded naturally from the leaf
surfaces that may have been previously punctured by the
insects; such droplets of semidry phloem sap were also
collected.
Chemical Analysis of Metabolites
To extract solutes, the frozen samples of the chlorenchyma or water-storage parenchyma were pulverized together with dry ice, ground in 0.5 mL of methanol:chloroform:water (12:5:3, v/v), and extracted at 25°C with 5 mL
of distilled water. After the samples were heated to 95°C
for 3 min to inactivate enzymes and then centrifuged, the
decanted supernatant was passed through C18 sample
preparation cartridges (Alltech Associates, Deerfield, IL)
presaturated with distilled water to remove lipophilic materials; the eluate was further cleaned by passage through
a 0.2-mm nylon filter. The final filtrate was collected for
metabolite analysis. To measure starch content (Wang and
Nobel, 1996), the insoluble portions of tissue extract were
resuspended and then centrifuged three times with methanol:chloroform:water (12:5:3, v/v) and twice with distilled
water to remove pigments, lipids, and remaining solutes.
The remaining precipitates were mixed with 2 mL of distilled water and then heated at 100°C for 2 h to suspend the
starch, which was hydrolyzed with amyloglucosidase (EC
3.2.1.3) at 55°C overnight; the released Glc was quantified
using Glc oxidase (EC 1.1.3.4; Sturgeon, 1990). Soluble proteins in the chlorenchyma or water-storage parenchyma
were also extracted at 0 to 2°C, according to procedures
developed for O. ficus-indica (Wang and Nobel, 1996).
The metabolites in the dissected vascular tissues of
freeze-dried samples were extracted at 60°C with 0.2 mL of
HPLC grade water for 30 min. The supernatant extracted
from the sections was then passed through 50 mL of the C18
Phloem Transport of Fructans in a CAM Plant
sample-preparation cartridge material and a 0.2-mm nylon
filter. Soluble proteins in the vascular tissues were extracted at 0 to 2°C with 0.2 mL of 50 mm Mops-KOH (pH
7.5), 2 mm DTT, 2 mm EDTA, 20 mg mL21 polyvinylpolypyrrolidone (insoluble), 0.5% (v/v) Triton X-100, and 1 mm
PMSF (Wang and Nobel, 1996).
Oligofructans and other sugars were separated by HPLC
at 24.0 6 0.5°C using a Microsorb Amino column (Rainin
Instrument, Emeryville, CA) with acetonitrile:water (70:30,
v/v) as the mobile phase and were detected by differential
refractometry (Frehner et al., 1984), which can separate
oligofructans up to a DP of 8. Individual sugars and fructans were identified and quantified by comparing them
with known concentrations of Fru, Glc, Suc, 1-kestose, and
nystose (1-kestose and nystose were obtained from TCI
America, Portland, OR; other reagents were from Sigma)
and with published standard chromatograms (Cairns and
Pollock, 1988; Pollock and Lloyd, 1994). The malate concentration was quantified spectrophotometrically (Wang
and Nobel, 1996) using malic dehydrogenase (EC 1.1.1.37).
The soluble proteins were quantified by the method of
Bradford (1976) using BSA as a standard, with a slight
modification for microscale analysis. The osmolality of the
phloem sap collected under mineral oil was measured with
a vapor pressure osmometer (model 5500, Wescor, Logan,
UT), and the concentrations of sugars, oligofructans, and
malate of the phloem sap were determined as described
above.
Measurement of Enzyme Activities
To measure fructosyltransferase activity, 0.5 to 1.0 g of
frozen chlorenchyma or 50 mg of dissected vascular tissues
was ground at 0 to 2°C with 1 to 2 mL of 50 mm citric acid,
5 mm DTT, 5 mm ascorbic acid, 2 mm EDTA, and 1 mm
PMSF at pH 5.5 (Lüscher and Nelson, 1995). After the
sample was centrifuged, ammonium sulfate was added to
35% saturation to the decanted supernatant for 15 min to
precipitate proteins. After the sample was centrifuged a
second time, the supernatant was decanted and ammonium sulfate was added to 60% saturation for 20 min to
precipitate fructosyltransferases. Again, the sample was
centrifuged and the enzyme pellet was resuspended in 50
to 100 mL of the solution used for extraction and dialyzed
overnight at 2 to 4°C against 20 mm His (pH 5.5). After
dialysis, 20 mL of the dialyzed protein solution, free of
sugars, was transferred to microcentrifuge tubes containing 80 mL of 25 mm Mes (pH 5.5) and 125 mm Suc or 63 mm
1-kestose. The sample was incubated at 27°C for 2 to 3 h,
and the reaction was stopped by heating to 90°C for 3 min.
The sample was centrifuged again, proteins in the supernatant were removed with a membrane filter, and the
solution was injected onto an HPLC column to separate
and quantify mono- and oligosaccharides. The fructosyltransferase activity was calculated as the amount of oligofructans produced per minute per milligram of protein.
To measure the activity of fructan hydrolases, about 20
mg of semidry phloem exudate collected without using
mineral oil wells was dissolved in 20 mL of 50 mm Mes (pH
5.5) and dialyzed at 2 to 4°C overnight against the Mes
711
buffer. After dialysis, the desalted phloem exudate was
transferred to microcentrifuge tubes containing 80 mL of 50
mm Mes (pH 5.5) and 63 mm 1-kestose or nystose. After the
sample was incubated at 30°C for 3 h (Pontis, 1990), the
reaction was stopped by heating to 90°C for 3 min. The
sample was centrifuged, proteins in the supernatant were
removed by passage through a membrane filter, and the
solution was analyzed by HPLC to separate and quantify
mono- and oligosaccharides. The activity of fructan hydrolases was calculated as the amount of Fru, Suc, and
1-kestose released per minute per milligram of protein.
Suc synthase (EC 2.4.1.13) and acid and alkaline invertases (EC 3.2.1.26) in the chlorenchyma and the vascular
tissues were extracted with 5 mm DTT, 2 mm EDTA, 5 mg
mL21 BSA, and 1 mm PMSF, and their activities were
determined as for O. ficus-indica (Wang and Nobel, 1996).
For phloem sap, the enzyme activities were determined
after the sap was dialyzed against 50 mm Mops-KOH (pH
7.2) plus 2 mm leupeptin and 1 mm PMSF. The activity of
alkaline invertase was measured by quantifying the
amount of Glc released after 50 mL of the enzyme solution
(extracted from the chlorenchyma or the vascular tissues)
and dialyzed phloem sap was mixed with 100 mL of 50 mm
Mops-KOH (pH 7.5) plus 150 mm Suc at 25°C for 30 min.
All data are presented as means 6 se (n 5 4 plants, unless
specified otherwise).
RESULTS
Suc was the predominate sugar in the chlorenchyma of
mature green (source) leaves of A. deserti and only one
fructan occurred, neokestose (DP 3), which was barely
detectable (Fig. 1A). On a molar basis, Suc accounted for
66%, Glc plus Fru accounted for 32%, and neokestose accounted for only 2% of total soluble sugars in the chlorenchyma (Table I). Neokestose was also only barely detectable in the water-storage parenchyma (Fig. 1B). On a molar
basis, it accounted for only 3% of total soluble sugars for
the water-storage parenchyma, whereas Suc accounted for
44%. The concentrations of individual sugars, particularly
Suc, and the osmolality were lower in the water-storage
parenchyma than in the chlorenchyma; also, total soluble
sugars accounted for 22% of the osmolality in the chlorenchyma and 9% in the water-storage parenchyma (Table I).
At least four oligofructans occurred in the vascular tissues of mature leaves of A. deserti in addition to the sugars
Glc, Fru, and Suc (Fig. 1C). On a molar basis, neokestose,
1-kestose (DP 3), nystose (DP 4), and an unidentified pentofructan together accounted for 46% of total soluble sugars
in such tissues, almost twice as high as the Suc concentration (Table I). Total soluble sugars in the vascular tissues
accounted for 45% of the osmolality. These four oligofructans also occurred in the phloem sap of mature leaves (Fig.
1D), where they accounted for 19% of total soluble sugars;
Fru accounted for 24%, and Suc accounted for 53% (Table
I). Total soluble sugars in the phloem sap accounted for
about 78% of its osmolality. The concentration of Suc was
3.6 times lower, that of nystose was 4.9 times higher, and
that of pentofructan was 3.2 times higher in the vascular
tissues than in the phloem sap (Table I).
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Wang and Nobel
Plant Physiol. Vol. 116, 1998
reaching a minimum of 30 mm, and then increased to a
maximum of 247 mm at the end of the night (Fig. 2B).
The activity of fructosyltransferases was not detected in
the chlorenchyma but was substantial in the vascular tissues (Table II). On a total soluble protein basis, the activity
of fructan hydrolases was 18 times higher in the phloem
sap compared with that of fructosyltransferases in the
chlorenchyma. The total soluble protein content in mature
leaves averaged 0.097 6 0.010 mg g21 phloem sap (n 5 4
plants) compared with 5.40 6 0.02 mg g21 fresh weight for
the chlorenchyma (n 5 5 plants). Thus, on a fresh weight
basis, the fructan hydrolase activity was about 3 times
higher in the chlorenchyma than in the phloem sap, accounting for less than 5% of the Suc hydrolyzed in the
phloem. For the chlorenchyma, Suc synthase is the major
enzyme responsible for cytosolic Suc breakdown, because
its activity was about 3.5 times higher than that of acid
invertase plus alkaline invertase (Table II). No acid or
alkaline invertase activity was detected in the phloem sap.
DISCUSSION
Figure 1. HPLC profiles of mono- and oligosaccharides in the various tissues (A, chlorenchyma; B, water-storage parenchyma; and C,
vascular) and in the phloem sap (D) of mature leaves of A. deserti. F,
Fru; G, Glc; and S, Suc. Numbers indicate the DP: 3a, neokestose;
3b, 1-kestose; 4, nystose; and 5, an unidentified pentofructan.
Suc and malate concentrations changed diurnally in a
reciprocal manner. Specifically, the Suc concentration in
the chlorenchyma gradually increased during the daytime,
reaching a maximum of 112 mm at dusk, and then gradually decreasing to a minimum of 45 mm immediately after
darkness (Fig. 2A). The malate concentration in the chlorenchyma gradually decreased throughout the daytime,
Fructan biosynthesis was apparently restricted to the
vascular tissues in mature leaves of A. deserti. In particular,
fructans were virtually absent from the chlorenchyma and
the water-storage parenchyma but accumulated in the vascular tissues. Coincident with the high concentrations of
oligofructans in the vascular tissues was the substantial
activity of fructosyltransferases (responsible for fructan
biosynthesis), whereas fructosyltransferase activity was
not detected in the chlorenchyma. The fact that oligofructans accumulated only in the vascular tissues and were
nearly absent from both the chlorenchyma and the waterstorage parenchyma suggests that back diffusion of these
fructans from the vascular tissues (such as from phloem
parenchyma cells) to the photosynthetic cells is extremely
low despite a substantial concentration gradient, consistent
with the polymer-trapping hypothesis (Turgeon, 1991).
Suc was the major carbon source for nocturnal malate
production in mature leaves of A. deserti. The nocturnal
decrease of Suc was 66 mm, which sustained the nocturnal
malate production of 217 mm (equivalent to 54 mm Suc,
because one Suc can be used to synthesize four malates;
Carnal and Black, 1989). Nocturnal production of other
organic acids, such as citric acid (Kluge and Ting, 1978),
can also utilize Suc, suggesting that little extra Suc is avail-
Table I. Concentrations of mono- and oligosaccharides in different tissues and in the phloem sap of mature leaves of A. deserti
Samples were harvested 4 to 5 h after the beginning of the light period. Data are means 6 SE; n 5 4 plants.
Sample
Fru
Glc
Suc
Neokestose
1-Kestose
Nystose
Pentofructan
Osmolality
Chlorenchyma
Water-storage
parenchyma
Vascular tissuesb
Phloem sap
15 6 2
8.2 6 1.1
17 6 2
8.6 6 1.4
65 6 8
14 6 2
2.3 6 0.3
1.1 6 0.1
nda
nd
nd
nd
nd
nd
445 6 7
342 6 36
32 6 4
89 6 12
27 6 4
15 6 2
55 6 9
199 6 15
3.3 6 0.5
9.6 6 1.9
27 6 4
46 6 5
31 6 4
6.3 6 2.4
35 6 5
11 6 1
469 6 52
482 6 45
mM
a
mOsm
b
nd, Not detected.
Included vascular parenchyma cells, phloem parenchyma cells, sieve tubes, and companion cells, and was
contaminated with a small amount of chlorenchyma and water-storage parenchyma.
Phloem Transport of Fructans in a CAM Plant
Figure 2. Daily changes of Suc concentration (A) and malate concentration (B) in the chlorenchyma of A. deserti. Hatched bars indicate nighttime. The data are means 6 SE for n 5 4 plants.
able for fructan synthesis, consistent with the small amount
of fructans in the chlorenchyma.
The particular cell type in the vascular tissues that is
responsible for fructan biosynthesis in mature leaves of A.
deserti is unclear. In Turgeon’s polymer-trapping model the
sites for the synthesis of RFO in Coleus blumei and Cucurbita
pepo (dicotyledonous species using the C3 photosynthetic
pathway) are intermediary cells (specialized companion
cells with numerous plasmodesmata connected to the
bundle-sheath cells; Turgeon, 1991). For A. deserti, a monocot using the CAM pathway, intermediary cells as found in
dicots are unlikely. Moreover, for the minor veins of C.
blumei and C. pepo, the bundle-sheath cells are directly
associated with the intermediary cells (Turgeon et al., 1975;
Fisher, 1986), but no such bundle-sheath cells were observed in the vascular strands of A. deserti (N. Wang and
P.S. Nobel, unpublished observations). Therefore, the sites
for oligofructan synthesis and accumulation in mature
leaves of A. deserti may be phloem parenchyma cells or
other types of vascular cells to be identified in the future.
The fructans in the phloem sap of A. deserti were apparently not from contamination with microorganisms. When
the phloem sap of O. ficus-indica is contaminated with
microorganisms, the activity of invertases often lead to
equal amounts of Glc and Fru after Suc is hydrolyzed (N.
Wang and P.S. Nobel, unpublished observations). Also,
6-kestose, a common end product of acid invertase action
on oligofructans (Cairns, 1993; Lüscher and Nelson, 1995),
was virtually absent from the phloem sap of A. deserti,
consistent with the lack of invertase activity there. On a
whole-tissue basis, the Suc concentration in the chlorenchyma (mesophyll cells) averaged about 70 mm over 24 h.
713
The volume of cytosol in mesophyll cells is about 5% of the
total cell volume for C3 and CAM species (Lüttge et al.,
1982; Winter et al., 1994; Haritatos et al., 1996), and about
26% of the cell Suc pool is in the cytosol for Suc-storage
species (Winter et al., 1994) such as A. deserti, the mature
leaves of which contained virtually no starch (N. Wang and
P.S. Nobel, unpublished observations). Thus, the Suc concentration in the cytosol of mesophyll cells of mature
leaves of A. deserti could be about 350 mm, about 1.8 times
higher than in the phloem sap, similar to the 1.5 times
higher Suc concentration in the cytosol of mesophyll cells
in mature leaves of Cucumis melo than in their sieve tubes/
intermediary cells (Haritatos et al., 1996).
The vascular tissues of A. deserti collected with a fine
syringe needle were always contaminated with small
amounts of chlorenchyma and water-storage parenchyma
(which had little detectable oligofructans); therefore, the
concentration of oligofructans in individual phloem parenchyma cells or other types of vascular cells is higher than
the average measured concentration in the vascular tissues.
If we assume that the total soluble sugars in the phloem
parenchyma cells or other types of vascular cells also accounted for about 78% of the osmolality, just as for the
phloem sap, sugar concentrations in these cells can be
estimated from the sugar concentrations in the vascular
tissues by multiplying by 1.75 (78/45%). Based on such a
calculation, sugar concentration gradients along the
phloem-loading pathway from the chlorenchyma to the
sieve tubes/companion cells can be approximated. In particular, Suc, 1-kestose, nystose, a pentofructan, and Glc
could diffuse from the chlorenchyma or from phloem parenchyma cells or other types of vascular cells into the
sieve tubes/companion cells, whereas the situation for Fru
is unclear because the Fru concentration in the cytosol of
chlorenchyma cells in mature leaves of A. deserti is unknown. In any case, such findings are in contrast with those
for plants with apoplastic loading pathways, such as Zea
mays (Bush, 1993), in which Suc is the only sugar in the
phloem sap (Ohshima et al., 1990). However, such findings
are similar to those from plants with symplastic loading
pathways, including the CAM species Xerosicyos danguyi,
in which substantial amounts of oligosaccharides, such as
raffinose and stachyose, are present in the companion cells
in addition to Suc (Madore et al., 1988; van Bel, 1993).
Table II. Enzyme activities in various regions of mature leaves of
A. deserti
Data are means 6 SE; n 5 4 plants.
Enzyme
Sample
Activity
nmol mg
Fructosyltransferases Chlorenchyma
Vascular tissues
Fructan hydrolases
Phloem sap
Suc synthase
Chlorenchyma
Acid invertase
Chlorenchyma
Phloem sap
Alkaline invertase
Chlorenchyma
Phloem sap
a
nd, Not detected.
21
protein min21
nda
14.6 6 2.0
260 6 27
14.1 6 1.5
3.0 6 3.0
nd
1.1 6 1.0
nd
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Wang and Nobel
Therefore, like the cucurbit X. danguyi, A. deserti may use
a polymer-trapping mechanism (Turgeon, 1991) for symplastic loading of photosynthates in its mature leaves. Suc
in photosynthetic cells may diffuse via plasmodesmata to
vascular parenchyma cells and then to phloem parenchyma
cells or other types of vascular cells. After Suc enters such
cells, it may be converted by fructosyltransferases to
1-kestose and higher DP fructans such as nystose and
pentofructan. Because the channel size of plasmodesmata
between the vascular parenchyma and the phloem parenchyma cells or other types of vascular cells may pass Suc
but restrict the diffusion of oligofructans such as 1-kestose,
nystose, and a pentofructan, oligofructan concentrations
can increase in the phloem parenchyma cells or other types
of vascular cells. These oligofructans may then diffuse from
such cells to the sieve tubes/companion cells via plasmodesmata. To our knowledge, this provides the first evidence that oligofructans are synthesized and transported in
the phloem of mature leaves of higher plants. The polymertrapping mechanism that operates in dicotyledonous C3
species may also be valid for oligofructan transport in
monocotyledonous species such as A. deserti.
Received August 1, 1997; accepted October 30, 1997.
Copyright Clearance Center: 0032–0889/98/116/0709/06.
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