Download The Response of the Phosphate Uptake System and the Organic

Plant Cell Physiol. 42(11): 1253–1264 (2001)
JSPP © 2001
The Response of the Phosphate Uptake System and the Organic Acid
Exudation System to Phosphate Starvation in Sesbania rostrata
Toshihiro Aono 1, 3, Naoki Kanada 2, Ayako Ijima 2 and Hiroshi Oyaizu 2, 4
1
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo, 113-8657 Japan
2
Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo, 113-8657 Japan
;
It is well known that the Pi uptake system via the highaffinity Pi transporter and the organic acid exudation system via PEPC are enhanced in the roots of Pi-starved
plants. In this paper, we compared the expression of these
two systems in Sesbania rostrata, a leguminous plant, on
whose roots and stems it forms nodules. When S. rostrata
plants were transferred to a 0 mM Pi nutrient solution, the
expression of both the high-affinity Pi transporter and
PEPC was enhanced within 2 d. The enhancement of the
expression of the high-affinity Pi transporter genes and the
PEPC gene coordinated with the increases in the Pi uptake
rate and the PEPC activity, respectively. This suggests that
the expression of the high-affinity Pi transporters and
PEPC is regulated in part at the transcript level. Furthermore, we examined which of the environmental or the
endogenous Pi level regulates the expression of these two
systems. The Pi content in the 6-day-old plants decreased to
a lower level than that in the 15-day-old plants when grown
in a 30 mM Pi solution. At that time, the expression of the
high-affinity Pi transporters and PEPC was enhanced only
in the 6-day-old plants. Moreover, the Pi content in plants
forming many nodules on their stems decreased. The
expression of the high-affinity Pi transporters and PEPC
was then enhanced in the nodulated plants. These facts suggest that the expression of these two systems may be regulated by the Pi content in the plants, not by the Pi concentration in the soil.
Key words: Phosphate starvation — Phosphate transporter —
Phosphoenolpyruvte carboxylase (EC 4.1.1.31) — Sesbania
rostrata — Stem nodule.
Abbreviations: PEPC, phosphoenolpyruvte carboxylase; RACE,
rapid amplification of cDNA ends; RT-PCR, reverse transcription
PCR; cMDH, cytosolic malate dehydrogenase; CS, citrate synthase.
The nucleotide sequences reported in this paper have been submitted to EMBL/GenBank/DDBJ under accession numbers AJ286743
for SrPT1, AJ286744 for SrPT2, AJ286750 for SrPEPC, AJ295348 for
partial cMDH, and AB057662 for partial CS.
3
4
Introduction
Sesbania rostrata is a leguminous plant, which forms
nodules in the stems as well as in the roots, and can perform
nitrogen fixation in the stem nodules. S. rostrata rapidly grows
and produces a large biomass. S. rostrata can normally grow
even if it is applied with low-grade phosphate rock instead of Pi
fertilizer (Patcharapreecha et al. 1993). Based on these characteristics, S. rostrata has been used as a convenient green
manure (Patcharapreecha et al. 1993). The deficiency of Pi is
thought to be one of the limiting factors of nitrogen fixation,
and that leguminous plants require a relatively large amount of
Pi. There has been reported an apparent interaction between the
fertilization of Pi and nodulation (Bonetti et al. 1984). In soybeans, the supply of Pi contributes to the higher nitrogen fixating ability and nodule mass rather than to plant growth (Israel
1987). The nitrogen fixation in nodules was then reduced under
Pi-deficient conditions (Mullen et al. 1988).
In soils, the concentration of available Pi for plants is usually very low because most of the Pi combines with iron, aluminium and calcium, to form scarcely soluble compounds.
Plants overcome Pi starvation in various ways. Several plants,
particularly legumes, are known to exude organic acids, such as
citrate, malate and succinate, in response to Pi starvation (Hoffland et al. 1989, Ohwaki and Hirata 1992, Petersen and Bottger 1991, Johnson et al. 1996a, Johnson et al. 1996b, Imas et
al. 1997). Organic acids exuded from the plant roots act as chelators, and can dissolve scarcely soluble Pi compounds. Consequently, the concentration of available Pi for plants rises around
the roots. The enhanced exudation was explained by the
increased synthesis of organic acids. Organic acids are provided by non-photosynthetic carbon fixation via phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), and the
increased synthesis of organic acids in the Pi-deficient roots is
due to the increase in the PEPC activity (Johnson et al. 1996a).
The activity of non-photosynthetic PEPC in the roots is
enhanced by Pi starvation (Pilbeam et al. 1993, Johnson et al.
1994). In white lupin roots, the enhanced synthesis of organic
acids coincides with the elevated PEPC activity (Johnson et al.
1994).
As another strategy to acquire Pi, an increase in the Pi
Present address, National Institute of Livestock and Grassland Science, Nishinasuno, Tochigi, 329-2793 Japan.
Corresponding author: E-mail, [email protected]; Fax, +81-3-5841-5334.
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Response to P deficiency in Sesbania rostrata
Fig. 1 Dry weights of the shoots (A and B) and the roots (C and D) of S. rostrata grown under various Pi conditions (´, 0 mM; open triangle,
30 mM; open circle, 50 mM; closed circle, 1 mM; open diamond, FePO4). Plants were transferred to the nutrient solution containing the indicated
levels of Pi at 6 d (A and C) or 15 d (B and D) after germination, and harvested every 2 or 3 d. Bars indicate standard deviation of mean (n = 3).
uptake rate in response to Pi starvation has been observed in
roots and cultured cells (Drew et al. 1984, Katz et al. 1986). Pi
is acquired by plants in an energy-mediated co-transport process driven by a proton gradient generated by the plasma membrane H+-ATPases (Sakano 1990). A dual mechanism model
for the uptake of Pi has been proposed (Furihata et al. 1992).
This is composed of two kinetically different uptake systems; a
high-affinity transport system and a low-affinity transport system. As most soils are deficient in Pi, the high-affinity transport system is thought to be primarily functional in the roots.
cDNAs for high-affinity Pi transporter genes have been
cloned and characterized from yeast (Bun-ya et al. 1991), fungi
(Harrison and van Buuren 1995, Versaw 1995), and plants
(Muchhal et al. 1996, Leggewie et al. 1997, Kai et al. 1997,
Smith et al. 1997, Liu, C. et al. 1998, Liu, H. et al. 1998).
These plant genes are primarily expressed in the roots and their
expression is induced under Pi-starved conditions.
In yeast, the expressions of several genes associated with
the pho-regulon, which responds to changes in the Pi concentration, are controlled at the transcriptional level. Yeast phoregulon includes the expression of the genes coding structural
proteins such as phosphatases and a high-affinity Pi trans-
porter, and positive and negative regulatory proteins for structural proteins (Lemire et al. 1985, Toh-e et al. 1988, Bun-ya et
al. 1991, Oshima et al. 1996, Lenburg and O’Shea 1996).
Recent studies have shown that Pi starvation in plants results in
the activation of multiple genes, suggesting that plants may
have a gene regulation system similar to the yeast pho-regulon.
In this study, we compared the regulation of the Pi uptake
system and that of the organic acids exudation system both
physiologically and molecular biologically in S. rostrata, to
determine whether or not these two systems are similarly controlled by the Pi condition. Furthermore, we analyzed the
expression of these two systems in the roots of plants consuming a large amount of Pi by forming many nodules on the stems
to determine if these two systems respond to the Pi content in
plants, and not to the Pi concentration in the soil.
Results
Effect of Pi starvation on growth of S. rostrata plants
The 6-day-old and the 15-day-old plants were transferred
to the solutions with different Pi levels, and grown in the
respective solutions. Fig. 1 shows the average dry weight of the
Response to P deficiency in Sesbania rostrata
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Fig. 2 Pi content in the roots (A and B) and in the shoot apices and the youngest leaves (C and D) of S. rostrata grown under various Pi conditions (´, 0 mM; open triangle, 30 mM; open circle, 50 mM; closed circle, 1 mM; open triangle, FePO4). Plants were transferred to the nutrient solution containing the indicated levels of Pi at 6 d (A and C) or 15 d (B and D) after germination. Bars indicate standard deviation of mean (n = 3).
plants grown in each solution. The plants grown under the
1 mM, 50 mM, and 30 mM Pi conditions grew similarly. However, there was a slight difference between the 6-day-old plants
under the 30 mM Pi and the 50 mM Pi conditions. The growth of
the plants under the 0 mM Pi conditions was suppressed. The
growth of the plants provided with FePO4 was slightly suppressed, but the dry weight continuously increased. For the 6day-old plants, the difference was not significant between the
plant growth in the 0 mM Pi solution and the other solutions till
6 d after transplanting. For the 15-day-old plants, the difference was not significant till 12 d after transplanting. The Pi
deficiency in the nutrient solution influenced the shoot growth
rather than the root growth. During the experiment, the Pi level
in the solution containing FePO4 was always under 1 mM.
Change of the Pi content in tissues under Pi-deficient conditions
The 6-day-old and the 15-day-old plants were transferred
to the solutions with different Pi levels, and grown. For both the
6-day-old and the 15-day-old plants, the Pi content in the roots
of the plants grown under the FePO4 and the 0 mM Pi conditions decreased within 1 or 2 d after transplanting, and the Pi
content in the roots under the 1 mM Pi and the 50 mM Pi condi-
tions remained unchanged (Fig. 2A, B). For the 6-day-old
plants, the Pi content in the roots of the plants grown under the
30 mM Pi conditions decreased to the same level as under the
0 mM Pi conditions. For the 15-day-old plants, the Pi content in
the roots of the plants grown under the 30 mM Pi conditions
somewhat decreased, but did not reach the level of the 0 mM Pi
conditions. In the stems and old leaves, there were similar tendencies as in the roots (data not shown). The Pi content in the
shoot apices and the youngest leaves of the plants grown only
under the 0 mM Pi conditions decreased (Fig. 2C, D). For the 6day-old plant, there was no significant difference between the
0 mM Pi conditions and the other conditions till 4 d after transplanting, and for the 15-day-old plants, 10 d after transplanting.
Enhancement of Pi uptake in response to Pi starvation
Plants were grown in the 0 mM Pi or 1 mM Pi solution for
4 d, and the initial uptake rates were measured over various Pi
concentration ranges of uptake solution (Fig. 3A, B). An analysis of the Pi uptake rate is usually performed by measurement
of the amount of Pi absorbed into the plants, using 32P or 33P.
This method enables an analysis over a wide range of Pi concentration, but non-specific adsorption and influx may cause
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Response to P deficiency in Sesbania rostrata
Fig. 3 Initial Pi uptake rates of S. rostrata over the 0–50 mM Pi concentration range. Six-day-old (A) or 15-day-old (B) plants were transferred to
the nutrient solution containing 1 mM (+) or 0 mM (–) Pi, and grown for 4 d. The plants were then transferred to the 32P-labeled nutrient solution
containing 0–50 mM Pi. After uptake time for 15 min, the decrease in the Pi concentration of the solution was measured. Bars indicate standard
deviation of mean (n = 3).
inaccurate estimation. Therefore, we analyzed the Pi uptake
rate by measuring the decrease in the Pi concentration in the
uptake solution. However, in the latter method, measurement of
the Pi uptake rate at a high Pi concentration cannot give accurate results. For that reason, we used concentrations from 0 to
50 mM Pi. Under such conditions, there were high-affinity and
low-affinity Pi uptake systems (Fig. 3A, B). From the shapes of
the curves, the Km for the high-affinity system was estimated to
be about 7 mM, and that for low-affinity one was 20–30 mM.
The Pi uptake rate of the Pi-starved plants was much higher
than that of the Pi-provided plants if the Pi concentration in the
solution was 20–30 mM or less. In the concentration range
higher than 12.5–15 mM, the Pi uptake rate of the Pi-provided
plants increased more steeply than that of the Pi-starved plants
with an increase in the Pi concentration. Moreover, for the Piprovided plants, the increase in the Pi uptake rate of the 15-dayold plants was steeper than that of the 6-day-old plants in this
concentration range. For the 6-day-old plants, the Pi uptake rate
in the Pi-starved plants was 3-fold higher than that of the Piprovided plants at 10 mM, and 2.5-fold at 20 mM. For the 15day-old plants, the Pi uptake rate in the Pi-starved plants was 5fold higher than that of the Pi-provided plants at 10 mM, and
1.5-fold at 20 mM.
Fig. 4 Amount of the exuded organic acids (A) and in vitro activity
of PEPC (B) in the roots of S. rostrata. Plants were grown in the solution containing 1 mM (+) or 0 mM (–) Pi for 4 d. After each treatment,
the organic acids exuded from the roots were collected over a 12 h
period, and determined by HPLC. The PEPC activity was determined
spectrophotometically at A340 for the roots of the plants. Bars indicate
standard deviation of mean (n = 3).
Enhancement of exudation of organic acids and PEPC activity
in response to Pi starvation
Plants grown in the 0 mM Pi or 1 mM Pi solution for 4 d
were transferred to the solution which was composed of
3.5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2, and the amount
of organic acids exuded from the roots for a period of 12 h was
measured (Fig. 4A). Citrate, malate, succinate, and fumarate
were detected in the root exudates. Malate was the major
organic acid. In the Pi-deficient plants, the amount of exuded
organic acids increased 5-fold in the 6-day-old plants, and 10fold in the 15-day-old plants compared with the Pi-provided
Response to P deficiency in Sesbania rostrata
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Table 1
ditions
Initial Pi uptake rates (nmol h–1 (g FW)–1) in roots of plants grown under various Pi con-
Age
Pi
Duration of treatment
2d
4d
1d
6d
15 d
1 mM
50 mM
30 mM
FePO4
0 mM
1 mM
50 mM
30 mM
FePO4
0 mM
76.3±13.3
—
—
—
81.5±17.1
70.1±16.4
—
—
—
109.9±11.6
78.5±13.7
—
—
—
198.7±18.2
67.3±14.4
—
—
—
293.9±23.3
82.1±7.9
83.9±6.8
191.4±19.9
272.4±12.0
278.7±17.6
63.8±14.3
73.2±7.4
82.9±27.1
318.2±33.6
322.6±25.8
6d
74.6±12.1
—
—
—
262.8±22.3
70.0±10.1
—
—
—
327.6±34.2
Plants were grown in the nutrient solution containing 1 mM, 50 mM, 30 mM, or 0 mM Pi, or FePO4 instead of
soluble Pi for 1–6 d. Measurement of the Pi uptake rates was performed with the 10 mM Pi uptake solution.
Means ± SD of three independent replications are presented.
Table 2 In vitro PEPC activity (nmol NADH min–1 (mg protein)–1) in roots of plants grown
under various Pi conditions
Age
6d
15 d
Pi
1 mM
50 mM
30 mM
FePO4
0 mM
1 mM
50 mM
30 mM
FePO4
0 mM
Duration of treatment
4d
1d
2d
40±3
—
—
—
67±9
52±9
—
—
—
72±10
42±5
—
—
—
178±33
53±3
—
—
—
197±11
38±10
45±6
56±6
209±13
203±19
53±5
58±4
69±20
202±16
192±14
6d
37±5
—
—
—
196±21
46±6
—
—
—
217±27
Plants were grown in the nutrient solution containing 1 mM, 50 mM, 30 mM, or 0 mM Pi, or FePO4 instead of
soluble Pi for 1–6 d. Means ± SD of three independent replications are presented.
plants. We measured the PEPC activity in the roots of the
plants with the 0 mM Pi and the 1 mM Pi treatment for 4 d (Fig.
4B). In the Pi-deficient plants, the PEPC activity increased 5fold in the 6-day-old plants, and 3-fold in the 15-day-old plants
compared with the Pi-provided plants.
Effect of various Pi conditions on Pi uptake and PEPC activity
in roots
To investigate the effect of Pi starvation duration on the Pi
uptake and the PEPC activity, plants were subjected to no Pi
treatment for 1 d to 6 d. Measurement of the Pi uptake rate was
performed with the 10 mM Pi uptake solution to avoid any
influence by the low-affinity uptake system (see Discussion).
The Pi uptake rate increased within 2 d of Pi starvation in the 6-
day-old plants, and within 1 d in the 15-day-old plants (Table
1). The Pi uptake rate continued to increase with increasing
duration of Pi starvation. The PEPC activity did not increase
within 1 d of Pi starvation. However, the PEPC activity
increased within 2 d of Pi starvation in both the 6-day-old and
the 15-day-old plants (Table 2). To investigate the effect of the
Pi level on the Pi uptake and the PEPC activity, plants were
treated with different Pi conditions for 4 d. The Pi uptake rate
was enhanced by the 30 mM Pi treatment in the 6-day-old
plants, but not in the 15-day-old plants (Table 1). The PEPC
activity was not significantly enhanced within 4 d of the 30 mM
Pi treatment in neither the 6-day-old nor the 15-day-old plants
(Table 2). However, the PEPC activity in the 6-day-old plants
was enhanced when plants were grown under the 30 mM Pi
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Response to P deficiency in Sesbania rostrata
Fig. 5 In vitro PEPC activity in the roots of S. rostrata grown in
30 mM Pi nutrient solution. Plants were transferred to the nutrient solution containing 30 mM Pi at 6 d or 15 d after germination, and harvested every 2 d. Bars indicate standard deviation of mean (n = 3).
conditions for more than 6 d, though that in the 15-day-old
plants was not (Fig. 5). When grown for 4 d under the 50 mM
Pi conditions, neither the Pi uptake rate nor the PEPC activity
was enhanced in both the 6-day-old and the 15-day-old plants
(Tables 1 and 2). Even when grown for more than 6 d under the
50 mM Pi conditions, both the Pi uptake rate and the PEPC
activity were not enhanced (data not shown).
Effect of stem nodules on Pi uptake and PEPC activity
Twelve-day-old plants were inoculated with Azorhizobium caulinodans ORS571 (Dreyfus et al. 1988) on their
stems. On the third day after inoculation, at which stage the
formation of the stem nodules was not visible, all of the Pi
uptake rate, the PEPC activity and the Pi content in the roots
had no significant difference between the inoculated plants and
the uninoculated plants (Table 3). On the ninth day after inoculation, there were about 40 to 60 stem nodules per plant (data
not shown). At this stage, both the Pi uptake rate and the PEPC
activity were enhanced in the roots of the plants forming stem
nodules (Table 3). Moreover, the Pi content in the roots (Table
Table 3
roots
Fig. 6 Northern blot analysis of the expression of the Pi transporter,
PEPC, cMDH, and CS genes in S. rostrata roots. Total RNA from
roots of the plants grown in the nutrient solution containing 1 mM (+)
or 0 mM (–) Pi for 4 d was hybridized with the labeled probes from
SrPT1, SrPT2, SrPEPC, S. rostrata cMDH, and S. rostrata CS. The
ethidium bromide-stained rRNA bands are shown as a loading control
at the bottom.
3), stems and leaves (data not shown) of the plants forming
stem nodules had decreased.
Effect of Pi starvation on the expression of SrPT1, SrPT2, and
SrPEPC genes
We cloned two kinds of full-length cDNAs coding the Pi
transporters (SrPT1, SrPT2) and a kind of cDNA coding PEPC
Effect of formation of stem nodules on Pi uptake rates, PEPC activity, and Pi content in
Days after initial inoculation
+ Inoculation
– Inoculation
3d
–1
9d
3d
9d
–1
65.9±15.1 295.4±35.4 64.3±15.0 70.1±16.8
Pi uptake rate (nmol h (g FW) )
174±17
50±9
61±7
PEPC activity (nmol NADH min–1 (mg protein)–1) 56±8
8.1±1.1
5.3±0.7
7.6±0.6
8.5±1.0
Pi content (mmol (g FW) –1) in roots
Plants were inoculated with A. caulinodans on the stems at 12 d after germination. The plants were then inoculated every 2 d on newly grown areas of the stems after the initial inoculation. Measurement of the Pi uptake
rates was performed with the 10 mM Pi uptake solution. Means ± SD of three independent replications are
presented.
Response to P deficiency in Sesbania rostrata
1259
Fig. 7 Expression of the SrPT1, SrPT2 and SrPEPC genes during Pi starvation. Total RNA was isolated from roots of plants grown in the nutrient solution containing 1 mM (+) or 0 mM (–) Pi for the indicated periods. The ethidium bromide-stained rRNA bands are shown as a loading control at the bottom.
(SrPEPC). The expression of the SrPT1, SrPT2, and SrPEPC
genes was compared between the Pi-starved and the Piprovided roots by Northern blot analysis (Fig. 6). Both the 3¢
gene specific probes for SrPT1 and SrPT2 hybridized to
approximately 2-kb transcripts. The 3¢ gene specific probe for
SrPEPC hybridized to approximately 3.4-kb transcripts. The
expression of these three genes was increased in the roots
under Pi-deficient conditions. The transcripts of the three genes
were detected more strongly in the 6-day-old plants than in the
15-day-old plants. In addition to the Pi transporters and PEPC
genes, we cloned partial cDNA fragments for the cytosolic
malate dehydrogenase (cMDH) and the citrate synthase (CS)
from the roots. The expression of the cMDH and CS genes was
examined by Northern blot analysis using the partial cDNAs as
probes (Fig. 6). The transcripts of the cMDH and CS genes had
little or no increase due to the Pi starvation for 4 d.
Sensitivity of SrPT1, SrPT2, and SrPEPC to Pi starvation
Plants were grown for 1 to 6 d under Pi-deficient and Pisufficient conditions. The expression of the SrPT1, SrPT2, and
SrPEPC genes had increased within 2–4 d of Pi starvation (Fig.
7). In the 15-day-old plants, the increase in the transcripts of
these genes was detected even within 1 d. To investigate the
expression of the SrPT1, SrPT2, and SrPEPC genes under different Pi conditions, plants were grown for 4 d in the solutions
with various Pi levels. The expression of the SrPT1 and SrPT2
genes was enhanced in the roots of the 6-day-old plants grown
under the 30 mM Pi and the FePO4 conditions as well as the
0 mM Pi conditions (Fig. 8). For the 15-day-old plants, an
increase in the transcripts of SrPT2 was detected in the roots
under the 30 mM Pi conditions, but such an increase in the transcripts of SrPT1 was not detected. The expression of the
SrPEPC gene was not enhanced in the plants under the 30 mM
Pi conditions irrespective of plant age.
Fig. 8 The expression of SrPT1, SrPT2 and SrPEPC genes under different Pi conditions. Total RNA was isolated from roots of plants
grown in the nutrient solution containing the indicated levels of Pi for
4 d. The ethidium bromide-stained rRNA bands are shown as a loading
control at the bottom.
Effect of stem nodules on the expression of SrPT1, SrPT2, and
SrPEPC genes
To investigate the effect of the formation of stem nodules,
the expression of the SrPT1, SrPT2, and SrPEPC genes in the
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Response to P deficiency in Sesbania rostrata
Fig. 9 The expression of SrPT1, SrPT2 and SrPEPC genes in roots of
S. rostrata forming stem nodules. Plants had been inoculated (+) or
uninoculated (–) with A. caulinodans ORS571 on stems every 2 d
since 12 d after germination. At 9 d after initial inoculation, total RNA
was isolated from roots. The ethidium bromide-stained rRNA bands
are shown as a loading control at the bottom.
roots of the plants inoculated or uninoculated with A. caulinodans ORS571 on the stems was examined (Fig. 9). The formation of stem nodules enhanced the expression of these genes
in the roots as did Pi starvation.
Discussion
To begin with, we examined the sensitivity of S. rostrata
to Pi starvation. For the 6-day-old S. rostrata plants, a significant difference in the growth was found between the plants
grown under the 0 mM Pi and the 1 mM Pi conditions at 6 d
after transplanting (Fig. 1A). Ozawa et al. (1995) and Neumann
et al. (1999) showed that the growth of white lupin, which is
tolerant to Pi starvation, was inhibited after 19 to 23 d of Pi
starvation. Generally, the sensitivity of the plants that require a
large amount of Pi to Pi starvation is relatively high under
hydroponic conditions. If such plants are grown under Pideficient conditions, the growth of the plants tends to be inhibited at an early stage. From these things, S. rostrata is seemingly sensitive to Pi starvation. However, the growth of S.
rostrata plants provided with FePO4 was not completely inhibited. As described in the results, the Pi level in the solution containing FePO4 was under 1 mM. This suggests that the growth
of S. rostrata is stopped only when the supply of Pi is completely severed, and this plant continues growing if the source
of Pi is given even when it is small. It follows that S. rostrata is
rather tolerant to Pi starvation.
When S. rostrata plants were transferred to Pi-deficient
conditions, such as the 0 mM Pi and the FePO4 conditions, the
Pi content in the tissues except for the growing points rapidly
decreased (Fig. 2A, B). However, the growth of S. rostrata
under the 0 mM Pi conditions did not quickly stop, and the
plants could grow for a while until the Pi content in the shoot
apices and the youngest leaves began to decrease (Fig. 1, 2).
For the plants under the FePO4 conditions, the Pi content in the
shoot apices and the youngest leaves remained at a high level,
and the growth of the plants did not stop. These facts suggest
that S. rostrata plants can easily translocate endogenous Pi, and
grow until the endogenous Pi has been consumed.
Based on the cases described below, it was found that the
response was different depending on both the environmental Pi
concentration and the plant age. First, the Pi uptake rate measured at 30 mM Pi was less than that at 50 mM Pi, and the Pi
uptake rate of the 6-day-old plants measured at 30 mM Pi was
less than that of the 15-day-old plants (Fig. 3A, B). Second,
there was a slight difference in the growth between the 6-dayold plants under the 30 mM Pi and the 50 mM Pi conditions, but
there was no difference in the growth of 15-day-old plants (Fig.
1, B). Third, the Pi content in the roots of the plants grown
under the 30 mM Pi conditions was lower than that of the plants
grown under the 50 mM Pi conditions, and the Pi content in the
roots of the 6-day-old plants was lower than that of the 15-dayold plants under the 30 mM Pi conditions (Fig. 2A, B). These
results suggest that the capacity of Pi uptake of S. rostrata is
developmentally different, and that this causes the differences
in the growth and the Pi content in the plants under 30 mM Pi
conditions between the 6-day-old and the 15-day-old plants.
We measured the initial Pi uptake rates over the 0 to
50 mM Pi range. Fig. 3A and B show the existence of two kinds
of uptake systems, the high-affinity and low-affinity systems,
even under low Pi conditions (0–50 mM). The Pi uptake rates of
the Pi-provided plants mainly reflect the high-affinity system
up to about 12.5–15 mM Pi, and the low-affinity system at more
than 15 mM Pi. The Pi uptake rates of the Pi-deficient plants
reflect the high-affinity system up to about 25 mM Pi, and the
low-affinity system at more than 25 mM Pi. Thus, when examining the capacity depending on only the high-affinity uptake
system, it is necessary to use the solution with very low Pi concentrations such as 10 mM Pi.
The Km for the high-affinity system is estimated to be
about 7 mM. This value is reasonable as the Km for the highaffinity system, as compared with other plants (Mimura 1999).
The Km for the low-affinity system seems to be very low. From
Fig. 3A and B, the deduced Km value for the low-affinity system is between 20 and 30 mM, which is much lower than many
other plants (Mimura 1999). However, some plants are known
to have low Km values for the low-affinity system. For example, the Km for the low-affinity system of the non-proteoid roots
of white lupin is 30.7 mM (Neumann et al. 1999), which is a
low value like that of S. rostrata. Because having such a low
Km, the low-affinity system is thought to affect the Pi uptake
rate in the low Pi concentration range (0–50 mM). In addition,
the influence of the low-affinity system on the Pi uptake rates is
larger in the 15-day-old plants than in the 6-day-old plants.
That is, the influence of the low-affinity system is developmentally different. It follows that the capacity of the Pi uptake
Response to P deficiency in Sesbania rostrata
changes with the growth stages. Meanwhile, the high-affinity
system was enhanced by Pi starvation in the S. rostrata roots as
well as in the barley roots (Drew et al. 1984) and the Catharanthus roseus protoplast (Furihata et al. 1992). Therefore, the
low-affinity system has a larger effect on the Pi uptake rates of
the Pi-provided plants than that of the Pi-starved plants because
the high-affinity system is not induced in the Pi-provided
plants. Based on these results, it can be said that the influence
of the low-affinity and the high-affinity systems on the Pi
uptake is determined by both the growth stage and the existence of Pi.
Many research groups have reported that the exudation of
organic acids from roots is enhanced under Pi-deficient conditions (Hoffland et al. 1989, Ohwaki and Hirata 1992, Petersen
and Bottger 1991, Johnson et al. 1996a, Johnson et al. 1996b,
Imas et al. 1997), and that the increased synthesis of organic
acids is due to an increase in the PEPC activity (Johnson et al.
1996a). In S. rostrata, both the exudation of organic acids from
the roots and the PEPC activity in the roots were enhanced by
Pi starvation (Fig. 4). In addition, malate was the predominant
organic acid exuded from the S. rostrata roots, and the exudation of citrate was only slightly enhanced. Takita et al. (1999)
demonstrated by using Al-phosphate utilizing carrot cells that
the CS activity increased during the excretion of citrate. These
results suggest that the carboxylation of phosphoenolpyruvate
to oxaloacetate by PEPC is enhanced in the S. rostrata roots
under Pi-deficient conditions, but that the conversion of oxaloacetate to citrate by CS is not. Actually, the expression of the CS
gene was not enhanced by Pi starvation in S. rostrata (Fig. 6).
However, Neumann et al. (1999) showed that the CS activity
was not increased though the exudation of citrate from the proteoid roots of the white lupin was increased. They also proposed that the exudation of citrate from the proteoid roots was
due to the increased biosynthesis and to decreased metabolization of citrate in the tricarboxylic acid cycle. Based on these
results, it cannot necessarily be said that the little increase in
the exudation of citrate is due to no increase in the CS activity.
We hypothesized that the enhancement of the conversion of
oxaloacetate to malate by cMDH caused the increase in the
exudation of malate. However, the expression of the cMDH
gene was only slightly enhanced by Pi starvation (Fig. 6),
though the activity of cMDH was not analyzed. A further analysis to determine the activity of the enzymes contained in the
organic acid metabolism is required to understand why the
malate exudation was enhanced by Pi starvation but citrate exudation was not.
In S. rostrata roots, both the transcripts of the Pi transporter genes and the PEPC gene were increased by Pi starvation. The level of the expression of the Pi transporter genes and
the PEPC gene (Fig. 7, 8) coordinated with the level of the Pi
uptake rate and the PEPC activity (Tables 1, 2), respectively. In
tomato, the increase in the transcripts of the Pi transporter
under Pi-deficient conditions coincides with the increase in the
transporter proteins and with the enhancement of the Pi uptake,
1261
so it is considered that the Pi uptake in tomato roots is under
transcriptional regulation of the Pi transporter genes (Muchhal
and Raghothama 1999). Because the increase in the transcripts
of SrPT1 and SrPT2 coordinates with the enhanced Pi uptake,
the expression of the Pi transporters in S. rostrata seems to be,
in part, under transcriptional regulation, as well as in tomato.
Generally, the expression of plant PEPC is regulated by transcription, posttranslational phosphorylation, oligomerization,
and protein turnover (Lepiniec et al. 1994). In the proteoid
roots of white lupin under Pi-deficient conditions, the increase
in the PEPC activity coincides with the increase in the PEPC
transcripts and amount of the PEPC proteins, and this fact
shows that the expression of PEPC in response to Pi starvation
is, in part, under transcriptional regulation (Johnson et al.
1996b). In S. rostrata, coordination of the increased expression
of the SrPEPC gene with the increase in the synthesis of
organic acids and the PEPC activity suggests that the expression of PEPC is, in part, under transcriptional regulation, as
well as in the proteoid roots of white lupin. The expression of
the Pi transporters and PEPC may be also under regulatory systems other than transcriptional regulation. However, there is no
doubt that Pi starvation in S. rostrata results in the transcriptional activation of the genes encoding the Pi transporters and
PEPC. It is necessary to further examine whether or not these
genes belong to the regulatory system like the pho-regulon in
S. cerevisiae.
We examined the response of the Pi uptake system and the
organic acid exudation system to Pi starvation. When plants
were transferred to the 0 mM Pi solution, both the Pi uptake rate
and the PEPC activity were enhanced by Pi starvation within
2 d (Tables 1, 2). The Pi content in the roots then reached a low
level within 2 d in the Pi-starved plants (Fig. 2). When the 6day-old plants were transferred to the 30 mM Pi solution, the Pi
uptake rate was enhanced within 4 d (Table 1), and the PEPC
activity was enhanced within 6 d (Fig. 5). Nevertheless, neither of them in the 15-day-old plants was enhanced under the
30 mM Pi conditions (Table 1 and Fig. 5). At that time, the Pi
content in the roots of the 6-day-old plants reached a low level,
but that of the 15-day-old plants did not (Fig. 2). These facts
suggest that the Pi uptake system and the organic acid exudation system may respond to the Pi level in plant tissues, not to
the environmental Pi level. If these systems respond to the
environmental Pi level, the Pi uptake rate and the PEPC activity of the 15-day-old plants should be enhanced under the
30 mM Pi conditions as well as that of the 6-day-old plants. Liu,
C. et al. (1998) reported a similar suggestion from the dividedroots experiment.
The Pi content in the root of the plants forming stem nodules decreased (Table 3). This decrease is likely because the
consumption of Pi in the nodules overtakes the Pi uptake in the
roots. Nitrogen fixation requires a large amount of Pi (Israel
1987). Kaur et al. (1999) presumed that during the active nitrogen fixation, the requirement of ATP is high. In addition to the
decrease in the Pi content in the roots, both the Pi uptake rate
1262
Response to P deficiency in Sesbania rostrata
and the PEPC activity were enhanced in the roots of the plants
forming stem nodules (Table 3). These facts confirm the
assumption that the Pi uptake rate and the PEPC activity was
enhanced by the low Pi content in the plants. It is remarkable
that nodulation enhances the Pi uptake system and the organic
acid exudation system. The enhancement of these two systems
will compensate S. rostrata for the Pi consumed by nitrogen
fixation.
In this study, we found the following characteristics of S.
rostrata. First, the growth of S. rostrata does not stop even if
the source of Pi is low. Second, both the Pi uptake system and
the organic acid exudation system are rapidly enhanced in
response to the decrease in the endogenous Pi content. Third,
these two systems are also enhanced by nodulation as well as Pi
starvation. Based on these results, it would not be an exaggeration to say that S. rostrata is adapted to Pi deficiency, and has
the mechanism to prevent nitrogen fixation from being suppressed by a lack of Pi.
Materials and Methods
Growth of plants
S. rostrata plants were grown in a chamber at 30°C under 12-/12h day/night cycles. Seeds were surface-sterilized and germinated in
vermiculite moistened with distilled water. Three d after germination,
the seedlings were transferred to the complete nutrient solution, and
then grown until used in the experiments. The complete nutrient solution was adjusted to pH 6.0 and contained 2.5 mM KNO3, 2 mM
Ca(NO3)2, 0.5 mM NH4NO3, 1 mM KH2PO4, 1.0 mM MgSO4, 0.5 mM
CaCl2, 50 mM Fe-EDTA, 50 mM H3BO3, 20 mM MnSO4, 3 mM ZnSO4,
0.05 mM Na2MoO4, 0.12 mM CuSO4, and 0.1 mM CoCl2. For the
experiments, 6- and 15-day-old plants were transferred from the complete solution to the nutrient solutions containing 0 mM, 30 mM,
50 mM, or 1 mM KH2PO4, or to the solution containing no KH2PO4
but supplied with FePO4. In the nutrient solutions of 0, 30, 50 mM Pi,
KCl was added instead of KH2PO4. In the nutrient solution supplied
with FePO4, 50 mg FePO4·nH2O was added per liter of solution. For
the experiments concerning the plant growth and the Pi content in the
tissues, all plants were grown in 4 liters of nutrient solution per plant
after transplanting. For the other experiments, the 6-day-old plants
were grown in 400 ml of nutrient solution per plant, and the 15-dayold plants were grown 4 liters of nutrient solution per plant. The nutrient solution was renewed every 2–3 d until the plants were used for the
experiments, then everyday after starting the experiment. The nutrient
solutions were continuously aerated.
Nodulation on stems
Twelve-day-old plants were inoculated with a culture of A.
caulinodans ORS571 (Dreyfus et al. 1988) on the stems. The plants
were then inoculated every 2 d on additionally grown areas of the
stems. All plants were grown in 4 liters of 1 mM Pi nutrient solution
per plant. The nutrient solution was renewed every 2–3 d before the
first inoculation, then everyday after starting inoculation.
Pi content in tissues
Plant tissues were frozen with liquid N2 and immediately thawed.
To extract the soluble Pi, each sample was boiled in hot water. The
extract was centrifuged at 5,000´g, and then the supernatant was filtered through a 0.45 mm filter. The Pi concentration was measured by
the molybdate-blue method (Murphy and Riley 1962).
Pi uptake
The roots of the plants were soaked in the nutrient solution deficient in Pi, then the plants were transferred to the 32P-labeled nutrient
solution containing 0–50 mM Pi. The composition of the other nutrients in the solution was the same as the complete nutrient solution.
The concentration of 32P was 370 MBq liter–1. For the 6-day-old
plants, the plants were grouped into ten batches and transferred to
20 ml of solution. For the 15-day-old plants, the plants were grouped
into three batches and transferred to 80 ml of solution. Fifteen min
after transfer, a 100 ml aliquot of sample was withdrawn from each
solution. The radioactivity of each sample was measured using a liquid scintillation counter (Aloka, LSC-5100). The initial radioactivity
of the labeled solution was also measured. The recorded cpm remaining in the solution was converted to the Pi concentration deduced from
the initial specific radioactivity. During the experiment, all solutions
were continuously aerated.
For the measurement of the initial Pi uptake rates of the plants
grown under various Pi conditions, the plants were transferred to the
32
P-labeled nutrient solution containing 10 mM Pi.
Analysis of exuded organic acids
After the treatments with various Pi levels, the plants were individually transferred to the test solution including K+, Ca2+, and Mg2+.
The 6-day-old plants were transferred to 40 ml of the test solution, and
the 15-day-old plants went into 400 ml of the solution. The solution
for exudation contained 3.5 mM of KCl, 2 mM of CaCl2, and 1 mM of
MgCl2, and the pH of the solution was adjusted to 6.3. Before transfer,
the roots were thoroughly washed with the test solution. Each plant
was grown with continuous light and aeration. After 12 h, each sample solution was syringe filtered (0.8 mm). An analysis of the organic
acids in the test solution was carried out according to Johnson et al.
(1996a) and Johnson et al. (1996b). The acidic fraction of each sample
was separated from the basic and neutral fractions by solid phase
extraction using a SAX column (SAX Bond Elut Jr, 500 mg, Varian).
Each column was attached to a syringe (50 ml), and preconditioned
with 4 ml of 100% methanol followed by 4 ml of 50% ethanol. The
sample was loaded onto the column, washed with 2 ml of 50% methanol, and then eluted with 4 ml of 2% HCl in methanol (acidic fraction).
The acidic fraction was dried and the residue was resuspended in
0.008 M H2SO4, and filtered (0.22 mm). The individual organic acids
were separated by a HPLC (Shimadzu, LC-6A) equipped with an
Aminex HPX-87H (300´7.8 mm) column (Bio-Rad) and an organic
acid guard column (Bio-Rad) with the eluent of 0.008 M H2SO4 at a
flow rate of 0.6 ml min–1 at 25°C. The organic acids were detected
with a UV detector (Shimadzu, SDP-6A) at 210 nm wavelength and
identified by comparing the retention times of the unknowns with
those of standard organic acid mixtures. To determine the average
recovery of each organic acid after solid phase extraction and HPLC,
organic acid mixtures with known concentrations were also extracted
and analyzed as mentioned above.
In vitro PEPC assay
Root tissue was frozen with liquid N2, and ground to a fine powder. The powder was homogenized in three volumes (w/v) of the
extraction buffer (50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM
DTT, 20% (w/v) glycerol, and 1% (w/v) serum albumin). The
homogenate was centrifuged for 10 min at 14,000´g, and the supernatant was used for the PEPC assay. The assay was performed in
100 mM Tris-HCl (pH 8.0), 10 mM KHCO3, 2 mM phosphoenolpyruvate, 2 mM MgCl2, 0.2 mM NADH, 2 units malate dehydrogenase,
and 30 ml extract in a total volume of 1 ml. The disappearance of
NADH was spectrophotometically monitored at A340. The protein concentration was determined using a protein assay kit (Bio-Rad).
Response to P deficiency in Sesbania rostrata
RNA isolation
Total RNA was isolated from the each tissue of S. rostrata plants
by phenol/SDS extraction and LiCl precipitation (Shirzadegan et al.
1991). Poly(A)+ RNA was isolated using Oligotex-dT30 (TAKARA).
RT-PCR (reverse transcription PCR) and cloning of PCR products
The RT-PCR method was used to obtain partial cDNA fragments
for all genes described in this report. The first strand cDNA was synthesized by reverse transcription of mRNA from Pi-starved roots using
oligo-dT12–18 primers (Amersham Pharmacia) and reverse transcriptase (Superscript II, Gibco BRL). Partial cDNA fragments for
each gene were amplified by PCR using the first strand cDNA as a
template and the appropriate primers. Amplification was carried out
using Taq DNA polymerase (Ampli Taq Gold, Perkin-Elmer). The
PCR products were ligated into T-vectors (pGEM-T easy vector,
Promega) for the subsequent analysis.
Cloning of cDNAs for Pi transporter genes
A pair of degenerate primers (sense primer, 5¢-TGG TT[C/T] III
[C/T]TI GA[C/T] AT[A/C/T] GCI TT[C/T] TA-3¢, and antisense
primer, 5¢-TT ICC III IGC IGC I[GA/CT] [A/G/T]AT ICC [A/G] TG3¢) was designed based on the amino acids sequences of the highly
conserved regions within the already known plant Pi transporters. Pi
transporter gene-specific fragments were amplified by the RT-PCR
method using these degenerate primers. A sequence analysis revealed
that two kinds of partial cDNA fragments for the Pi-transporter were
amplified by RT-PCR. These cDNA fragments were stretched by the 5¢
and 3¢ RACE (rapid amplification of cDNA ends) method using the
MARATHON cDNA cloning system (CLONTECH) with cDNAspecific primers (5¢ RACE primers, 5¢-GCC TTG CTGGGA ATA TCT
CTG CTG GG-3¢ and 5¢-GCC TTG CTG GGA AAA TCT CTG CTG
GC-3¢; 3¢ RACE primers, 5¢-AGT GCA ATT GGA TGG ATC CCT
CCT G-3¢ and 5¢-ACT GCC ATT GGA TGG ATC CCT CCT G-3¢)
according to the manufacturer’s recommendations, then full-length
cDNAs (SrPT1 and SrPT2) were obtained.
Cloning of cDNA for PEPC gene
The cloning of PEPC cDNA was also performed using the MARATHON cDNA cloning system. A partial cDNA fragment for PEPC
was obtained by the RT-PCR method using a pair of primers (sense
primer, 5¢-TCC TGA TGA TAA GCA GGA GCT-3¢, and antisense
primer, 5¢-ACA TCA GAI GGT GCA GTT CCC AT-3¢), which were
designed based on the amino acids sequences of the highly conserved
regions within the already known plant PEPC. This cDNA fragment
was stretched by 5¢ and 3¢ RACE using two cDNA-specific primers (5¢
RACE primer, 5¢-CAA GAA TTT CCC TAT GTC CAG CCA CC-3¢,
and 3¢RACE primer, 5¢-GAG TGG AAA ACG CCC TCT ATT CGG
ACC-3¢), then the full-length cDNA (SrPEPC) was obtained.
Cloning of partial cDNA fragments for cMDH and CS
A partial cDNA fragment for cMDH was amplified by the twostep PCR in accordance with Kirby (2000) using two pairs of degenerate primers (cMDHP1/cMDHP2 and cMDHP3/cMDHP4) which were
designed by Kirby (2000). A partial cDNA fragment for CS was also
amplified by the two-step PCR using a kind of sense primer (5¢-CA[C/
T] CCI ATG ACI CA[A/G] TT[C/T] GC-3¢) and two kinds of antisense primers (first step PCR, 5¢-TGI GC[A/G] TCI AC[A/G] TTI
GGC CA-3¢, and second step PCR, 5¢-GC[A/G] AA[C/T] TCI C[T/
G][C/T] TG[A/G] CA-3¢). The sense primer was used in both the firststep and the second-step PCR. A first-step PCR product was generated from the first strand cDNA. The second-step PCR was performed
using the diluted first-step PCR product as a template. The primers
used in the PCR were designed based on the amino acids sequences of
1263
the highly conserved regions within the already known plant CS.
Sequence analysis
A DNA sequence analysis was performed by the dideoxysequencing method with a DNA sequencer (model 310, Applied Biosystem). The sequences were analyzed using the software GENETYX
and the PROSITE data bank.
Northern blots
Ten mg of total RNA was electrophoretically separated on 1%
denaturing formaldehyde agarose gels and blotted onto nylon membranes (Sambrook et al. 1989). The nylon membranes were hybridized
overnight with 32P-labeled probes in a solution containing 50% formamide, 5´ Denhardt’s, 5´ SSPE, 0.1% (w/v) SDS, and 100 mg ml–1
denatured salmon-sperm DNA at 42°C. After hybridization, the blots
were washed twice in 2´ SSPE and 0.1% SDS at 65°C for 15 min,
once 1´ SSPE and 0.5% SDS at 65°C for 30 min, and once 0.1´ SSPE
and 0.1% SDS at room temperature for 15 min. The blots were
exposed to X-ray films between intensifying screens at –70°C for 16 h.
The gene specific probes for SrPT1, SrPT2 and SrPEPC were obtained
by PCR amplification of the 3¢-untranslated regions using the cDNA
clones as templates, and with gene specific primers.
Acknowledgements
The authors thank Prof. Satoshi Matsumoto for helpful suggestions and Keitaro Tanoi for excellent technical assistance with organic
acids analysis.
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(Received December 7, 2000; Accepted September 6, 2001)