Download Chemical Composition of Phloem Sap from the Uppermost Internode

Plant CellPhysiol. 31(2): 247-251 (1990)
JSPP © 1990
Chemical Composition of Phloem Sap from the Uppermost Internode
of the Rice Plant
Hiroaki Hayashi and Mitsuo Chino
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo,
Bunkyo-ku, Tokyo, 113 Japan
The chemical composition of phloem sap from the uppermost internode of rice plants (Oryza
sativa L., var. Kantou), one week after anthesis, was compared with that of phloem sap from the
leaf sheath of a young seedling. The pure phloem sap from rice plants was collected by an insect
laser technique.
The phloem sap from the uppermost internode contained a high level of sucrose (573.8 mM)
which was the only sugar detected. The concentrations of total amino acids, potassium and ATP
were 124.8 mM, 40.4 mM and 1.76 mM, respectively. The concentration of sucrose was three
times higher and the potassium level was one third as high in the internode sap as in the phloem
sap from the leaf sheath. The total concentration of amino acids was almost the same, but the
relative amount of each amino acid was quite different. The ratios of levels of Glu to Gin and of
levels of Asp to Asn in the phloem sap from the uppermost internode were smaller than those in
the phloem sap from the leaf sheath. The adenylate energy charge was 0.92-0.93 in both types of
phloem sap.
The amino acid composition of the phloem sap from the uppermost internode was compared
with that of the phloem sap of the flag leaf and the endosperm sap of the same plant, one week
after anthesis. The differences in composition along the phloem pathway suggest the selective
translocation of amino acid.
Key words: Amino acid — ATP — Phloem sap (rice) — Potassium — Sucrose — Uppermost
internode.
Since phloem transport plays a very important role of
the growth of sink organs, including grains and fruits, it is
important to define the chemical composition of phloem
sap at the loading site as well as at the unloading site. During transport from sources to sinks, it is assumed that the
chemical composition of phloem sap undergoes changes
due to the exchange of solutes between xylem, phloem and
surrounding tissues.
To clarify the mechanism of phloem transport and the
relationship between vegetative or reproductive growth and
phloem transport, phloem sap must be collected close to
the loading site and close to the unloading site. At the
loading site, the chemical composition of phloem sap
reflects the loading of sugars, amino acids and other compounds. At the unloading site, the chemical composition
of phloem sap reflect not only the loading processes but
also the exchange of solutes between phloem or xylem and
the surrounding cells along the transport pathway. The
phloem sap at the unloading site can supply nutrients for
the growth of grains or growing points.
Only a limited number of plants, such as wheat (Fisher
and Macnicol 1986, Fisher and Gifford 1986) and white
lupin (Pate et al. 1979), have been successfully used for collection of phloem sap along the pathway of phloem transport. In the case of wheat, phloem sap from aphid stylets
was collected along the transport pathway from the flag
leaf to the crease of the grain (Fisher and Gifford 1986).
Phloem sap from white lupin was collected from shallow incisions at the stem base, petioles, stem top and fruits (Pate
et al. 1979). The importance, in these species, of amides in
the phloem sap near the unloading site has been stressed
(Fisher and Macnicol 1986, Pate et al. 1979).
With respect to rice, phloem sap has been obtained only from the leaf sheath of seedlings (Kawabe et al. 1980,
Fukumorita and Chino 1982). In this study, the collection
of rice phloem sap from the stylets of brown planthoppers
Abbreviation: AEC, adenylate energy charge.
247
248
H. Hayashi and M. Chino
was achieved by severing the stylet with a laser beam. It
has proved physically impossible to aim the beam at stylets
of insects mounted on the uppermost internode of normal
varieties of rice because of the flexibility of the uppermost
internode. In our experiments, we selected a dwarf rice
with a thick uppermost internode, Oryza saliva L., var.
KANTOU, and phloem sap was successfully obtained from
the leaf sheath of the seedlings, from the leaf sheath of the
flag leaf and from the uppermost internode. The chemical
composition of these saps were compared in this study.
Materials and Methods
10
Plant materials—Rice plants (Oryza saliva L., var.
Kantou) were grown in a complete solution of nutrients
(Hayashi and Chino 1986a). Nitrate was included as the
only source of nitrogen. Plants were grown in halfstrength solution until the fourth-leaf stage. The nutrient
solution was renewed every 3 days and the pH was adjusted
to 5.5 daily. Plants were cultured under natural light in a
greenhouse at 28°C in the daytime and 23°C at night.
Collection of phloem sap and endosperm sap—Phloem sap was collected by the insect laser technique (Kawabe
et al. 1980). Adult' female brown planthoppers (Nilaparvata lugens Stal.) were used for this procedure. To collect
pure phloem sap, the beam of a YAG laser (NEC, Tokyo,
Japan) was focused on the stylet to sever it while the insect
was sucking the sap from the 6th-7th leaf sheath at the
7th- to 8th- leaf stage, from the leaf sheath of the flag leaf
one week after anthesis, and from the uppermost internode
one week after anthesis. The exudate was easily collected
by placing a microcapillary (Drummond Scientific Co.,
U.S.A.) over the cut end of the stylet using a micromanipulator. Endosperm sap was sampled using microcapillaries
after half of the endosperm was cut off with a razor. All
samples were collected in a sampling room (25°C, 240|iEm ~ 2 s ~ ' ) in the daytime. Each sample was diluted one
hundred-fold and stored at — 20°C until analysis.
Analysis—Sugar composition was determined by HPLC
(Hitachi Ltd., Tokyo, Japan) on a GL-C610 column
(Hitachi-Kasei Co., Tokyo, Japan) with a refractive
20
30
40 min
Fig. 1 Analysis by HPLC of nucleotides in the phloem sap the
uppermost internode.
Elution (1 ml/min) was carried out with
a linear gradient from 80% buffer A in buffer B to 50% buffer A
over the course of 45 min. Buffer A was 6% CH 3 CN. Buffer B
contained 0.36 M NH4C1, 0.06 M KH2PO4 and 0.06 M K2HPO4 in
6% CH3CN. X indicates an unidentified peak.
index monitor (Hitachi Ltd.) using 10 jA of diluted sample. Ion-exchanged water was used as eluant. The amino
acid composition was also analyzed on an HPLC system
for the the analysis of o-phthalaldehyde-amino acids with
10 fi\ of the diluted sample. A #2619-F column (Hitachi
Ltd.) and a fluoro-spectrophotometer (Hitachi Ltd.) were
used (Hayashi and Chino 1986a). Nucleotide composition
was determined on an HPLC system equipped with a
#3O13-N column and UV (260 nm) spectrophotometer
(Japan Spectroscopic Co., Tokyo, Japan). A typical chromatogram and the analytical conditions are shown in
Figure 1. Potassium concentrations were determined by
ion chromatography (Hayashi and Chino 1986a).
Results
Sampling of sap—The success rate for collection of
phloem sap from the uppermost internode was 2% of cutting trials. The rate of exudation from the severed stylets
was 2.0/il/h, on average, but the exudation did not con-
Table 1 Chemical Composition of rice phloem sap
Leaf sheath of seedling
(7th- to 8th- leaf stage)
Uppermost internode
(one week after anthesis)
(mM)
Sucrose
573.8 ±123.1
205.5 ±79.9
Total amino acids
124.8 ± 25.6
103.2 ±22.3
40.4 ± 19.9
147.1 ±42.5
Potassium
ATP
1.76±
0.16
Each value is the mean±SD of results from at least 5 replicates from different plants.
1.63± 0.18
Chemical composition of rice phloem sap
tinue for more than 2 h. It was very difficult to obtain the
phloem sap from the leaf sheath of flag leaves. Only one
sample (0.2 fil) was available for analysis. Endosperm sap
(0.2-0.4//I/grain) was collected from the grain at the middle part of the ear.
Composition of phloem sap from the uppermost internode and from the leaf sheath of seedlings—Table 1 and
Table 2 show the chemical composition of phloem sap collected from the uppermost internode, one week after anthesis, and from the leaf sheath of a seedling at the 7thto 8th- leaf stage. The concentration of sucrose in the
phloem sap from the uppermost internode was three times
that in the phloem sap of the leaf sheath of seedlings. Sucrose was the only sugar transported to the ear.
Although the total concentration of amino acids was
the same, the predominance of amides in the sap of the uppermost internode was remarkable. While Ser, Asn, Gin,
Lys and Arg accounted for 80.5% of total amino acids in
the phloem sap of the uppermost internode, Asp, Ser, Asn,
Glu and Gin made up 10.1% of the total amino acids in the
sap of seedlings. The concentration of potassium in the
sap of the uppermost internode was 40.4 mM which was
one third of that in the sap from the leaf sheath of seedlings. The nucleotide composition is summarized in Table
3. ATP was dominant in the rice phloem sap. Since levels
of AMP and ADP were negligible, the AEC was 0.92-0.93,
249
Table 3 Nucleotide composition of rice phloem sap
Uppermost internode
(one week after anthesis)
Leaf sheath of seedling
(7th-to 8th-leaf stage)
(mM)
AMP
ADP
CTP
UTP
ATP
GTP
AEC
trace
0.31 ±0.04
0.08 ±0.03
0.27±0.06
1.63±0.18
0.21 ±0.04
0.92±0.01
trace
0.27±0.04
0.04±0.01
0.10±0.02
1.76±0.16
0.22±0.04
0.93 ±0.01
Each value is the mean of results from 5 samples from different
plants.
a rather high value.
Amino acid compositions of phloem saps from flagleaf sheath and uppermost internode and of endosperm sap
—Table 4 shows the chemical composition in terms of
amino acids of phloem saps from the flag-leaf sheath and
uppermost internode and of the endosperm sap, one week
after anthesis. All saps were obtained from a single
plant. The proportions of amino acids in the sap from the
Table 4 Amino acid composition of rice phloem sap
along the transport pathway and endosperm sap
Table 2 Amino acid composition of rice phloem sap
Uppermost internode
(one week after anthesis)
(molar%)
Asp
Thr
Ser
Asn
Glu
Gin
Pro
Gly
Ala
Val
Cys
Met
Lie
Leu
Tyr
Phe
Lys
His
Arg
Flag-leaf sheath Uppermost internode Endosperm
sap
phloem sap
phloem sap
Leaf sheath of seedling
(7th- to 8th- leaf stage)
0.8
4.4
13.9
17.9
trace
30.8
1.4
1.0
0.6
3.6
trace
0.08
1.7
2.1
1.2
0.9
6.3
1.7
11.6
Samples were representative of each stage.
19.4
5.4
12.4
5.9
13.6
19.0
1.5
0.4
2.0
3.1
trace
0.3
2.5
2.8
1.8
1.8
3.0
1.1
4.1
Asp
Thr
Ser
Asn
Glu
Gin
Pro
Gly
Ala
Val
Cys
Met
He
Leu
Tyr
Phe
Lys
His
Arg
11.2
4.1
10.8
16.0
14.5
17.6
1.2
trace
1.7
4.0
trace
trace
1.9
1.6
0.5
0.4
4.8
0.9
8.9
(molar%)
1.4
4.6
7.8
12.4
0.8
42.0
trace
trace
0.2
2.9
trace
0.1
1.5
1.5
0.5
0.5
5.8
0.8
17.3
0.4
2.7
10.8
47.5
0.4
30.7
trace
4.2
trace
0.7
1.1
trace
0.2
0.2
trace
trace
0.3
0.7
0.1
All samples were obtained from a single plant, one week after anthesis.
250
H. Hayashi and M. Chino
leaf sheath of the flag leaf were generally similar to those in
the phloem sap from the leaf sheath of the seedling. The
ratio of Asn and Gin to total amino acids was the highest in
the endosperm sap, next highest in the uppermost internode sap and the lowest in sap of the flag-leaf sheath. Ser,
Asn and Gin accounted for 89% of the total amino acids in
the endosperm sap, while levels of Lys and Arg were negligible. These results suggest that Gin and Asn are important
as sources of nitrogen for formation of grain.
Discussion
An understanding of the changes in the chemical composition of phloem sap along the route from leaf to grain is
essential if we are to elucidate the mechanisms involved in
the unloading of the constituents of phloem or the contribution of these constituents to the formation of grain.
Sucrose was the only sugar transported through phloem to the ear and its concentration in the sap was found to
be about 570 mu. In white lupin, a significant gradient in
the concentration of sucrose through the stem toward the
fruits or vegetative apices was detected (Pate et al. 1979).
Demonstrable gradients of solutes occurred only between
the crease sieve tubes and the endosperm cavity, not along
the transport pathways from the peduncle to the crease of
the wheat ear, as reported by Fisher and Gifford (1986). In
the present experiments, no gradient in the concentration
of sucrose could be demonstrated along the transport pathway, because the amount of phloem sap from the flag-leaf
sheath was too small to allow analysis of the sucrose concentration. Therefore, we assume that the chemical composition of phloem sap from the flag-leaf sheath (loading
site), one week after anthesis, is about the same as that of
the phloem sap from the leaf sheath at the 7th- to 8th- leaf
stage.
Most phloem saps contain high concentrations of sucrose and potassium (Hall and Baker 1972, Pate et al. 1974,
Hocking 1980 and Fukumorita and Chino 1982). As
shown in Table 1, the concentration of potassium near the
unloading site was low compared to that at the loading
site. This tendency was also seen in the wheat phloem sap
(Hayashi and Chino 1986a, Fisher 1987). High levels of
potassium at the loading site suggest the importance of potassium for loading.
Not only ATP, but also ADP, UTP and GTP were
found in rice phloem sap and the AEC was high in the
sap. Gardner and Peel (1969) measured the level of ATP
in the willow phloem sap obtained through aphid stylets.
ATP in sieve tubes was the direct source of energy for
phloem loading of sucrose (Spanswick 1986). The high
AEC in sieve tubes reflects the rapid regeneration of ATP
in the sieve elements or companion cells, although the
mechanism involved in this process has not yet been clarified. Phloem sap from the uppermost internode of rice also
contained high levels of ATP. Although an energy-dependent and carrier-mediated transport process that controls unloading has been suggested (Throne and Rainbird
1983, Eschrich 1986), the unloading process in maize
pedicel tissue was not inhibited either by apoplastic pH or
by metabolic inhibitors. (Porter et al. 1985). It is clearly
necessary to study the relationship between the level of
ATP or the AEC and the unloading process.
The slight changes in the amino acid composition of
samples from the sieve tube and endosperm cavity of wheat
may demonstrate the independence of the amino acids in
sap from the amino acid metabolism in the surrounding
organs (Fisher and Macnicol 1986). However, in rice, the
higher concentrations of Gin and Asn in the sap from the
uppermost internode than in the leaf-sheath sap suggest
metabolic changes from Glu to Gin and from Asp to Asn
or the selective unloading of Asp and Glu and the exchange
of these amino acid between phloem and xylem on the way
from leaf to panicle. In rice plants supplied with nitrate,
Asp and Glu were dominant in the phloem sap from leaves
(Hayashi and Chino 1986a, Hayashi and Chino 1986b).
The observation that Gin and Asn were dominant in the
phloem sap near the unloading site, even when the rice
plants were supplied with nitrate, suggests the importance
of these amides in the transport of nitrogen to grains. The
importance of amides in the phloem sap close to the
unloading site was also noted in wheat (Fisher and Macnicol 1986) and in some legumes (Pate et al. 1979, Peoples
et al. 1985). Although Gin and Asn were also dominant in
the rice endosperm, the amino acid composition of the endosperm was characterized by a somewhat larger predominance of Cys and Gly and an absence of Arg and Lys.
Further studies of unloading of amino acids into the endosperm are needed, particularly in relation to protein synthesis.
This study was supported by a Grant-in-Aid for Scientific
Research, no. 63110003, from the Japanese Ministry of Education, Science and Culture.
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