The Plant Journal (2007) 50, 545–556
doi: 10.1111/j.1365-313X.2007.03070.x
Cell division activity determines the magnitude of phosphate
starvation responses in Arabidopsis
Fan Lai, Jennifer Thacker, Yuanyuan Li and Peter Doerner*
Institute of Molecular Plant Science, School of Biological Sciences, Daniel Rutherford Building, King’s Buildings, University of
Edinburgh, Edinburgh, EH9 3JH, UK
Received 31 July 2006; revised 29 November 2006; accepted 16 January 2007.
*
For correspondence (fax +44 131 650 5392; e-mail [email protected]).
Summary
Phosphate (Pi) is a major limiting factor for plant growth. Plants respond to limiting Pi supplies by inducing a
suite of adaptive responses comprising altered growth behaviour, enhanced Pi acquisition and reduced Pi
demand that together define a distinct physiological state. In Pi-starved plants, continued root growth is
required for Pi acquisition from new sources, yet meristem activity consumes Pi. Therefore, we analysed the
relationship between organ growth, phosphate starvation-responsive (PSR) gene expression and Pi content in
Arabidopsis thaliana under growth-promoting or inhibitory conditions. Induction of PSR gene expression
after transfer of plants to Pi-depleted conditions quantitatively reflects prior levels of Pi acquisition, and hence
is sensitive to the balance of Pi supply and demand. When plants are Pi-starved, enhanced root or shoot growth
exacerbates, whereas growth inhibition suppresses, Pi starvation responses, suggesting that the magnitude of
organ growth activity specifies the level of Pi demand. Inhibition of cell-cycle activity, but not of cell expansion
or cell growth, reduces Pi starvation-responsive gene expression. Thus, the level of cell-cycle activity specifies
the magnitude of Pi demand in Pi-starved plants. We propose that cell-cycle activity is the ultimate arbiter for Pi
demand in growing organs, and that other factors that influence levels of PSR gene expression do so by
affecting growth through modulation of meristem activity.
Keywords: phosphate starvation responses, gene expression, nutrient signalling, plant growth control, cell
division control.
Introduction
Phosphorus, which plants can only acquire as phosphate (Pi)
anion, is frequently the most limiting macro-nutrient for
plant growth (Marschner, 1995). Although Pi is widely distributed in soils, it is difficult for plants to assimilate for
several reasons. Pi solubility is strongly pH-dependent.
Furthermore, Pi is very tightly bound to soil minerals, and
hence mobility within soils is very low (Tinker and Nye,
2000). As a result, Pi distribution within soils is highly heterogeneous, even at very small spatial scales (Strawn et al.,
2002). Therefore, in global agriculture, phosphate seriously
limits plant growth and crop yields.
Plants respond to insufficient Pi nutrition by inducing a
syndrome of adaptive changes that lead to a distinct
physiological state involving altered growth behaviour,
enhanced Pi acquisition and reduced Pi demand. Enhanced
Pi uptake is facilitated by changes to root and to root hair
growth rates and patterns that increase surface area (Bates
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Journal compilation ª 2007 Blackwell Publishing Ltd
and Lynch, 1996, 2000a, 2000b; Ma et al., 2001; Williamson
et al., 2001). In concert with the secretion of organic acids,
nucleases and phosphatases that increase Pi mobility in the
soil and liberate Pi from organically bound phosphate (Abel
et al., 2002), these changes enhance the capacity for Pi
acquisition. Reduction of demand is promoted by the
substitution of phospholipids by sulfo- and galactolipids in
membranes (Hartel and Benning, 2000; Hartel et al., 2000; Yu
et al., 2002), while the concomitant hydrolysis of Pi from
phospholipids by phospholipases (Cruz-Ramı́rez et al., 2006;
Nakamura et al., 2005) mobilizes Pi from internal sources. Pi
demand is also reduced by induction of metabolic shunts
that minimize the requirement for Pi in basic metabolism
(Plaxton and Carswell, 1999). Together, such changes that
reduce Pi demand may be considered as minimizing the
immobile fraction of total cellular Pi in favour of mobile,
freely convertible forms (e.g. Pi or ATP) required for meta545
546 Fan Lai et al.
bolism and growth. Pi starvation also promotes re-distribution of Pi to sink tissues (Marschner et al., 1996). In Pi-replete
plants, large amounts of phosphate are stored in the
vacuoles of shoot tissues (Bieleski, 1973; Mimura, 1999),
which are preferentially transported to roots in Pi-depleted
conditions. Altered growth behaviour, augmented acquisition and reduction of demand in response to Pi limitation
correlate with extensive changes to gene expression patterns of phosphate starvation-responsive (PSR) genes
(Hammond et al., 2003; Misson et al., 2005; Wu et al., 2003).
Heterogeneously distributed mineral nutrients with lowmobility, such as phosphate or iron, are only acquired
efficiently by root systems with large surface areas, in which
the root surface is brought into direct contact with the soil
(Lynch, 1995). As a consequence, maintenance of root
growth is critical, even for plants that have acquired a
physiological state of Pi starvation, to increase the likelihood
of coming into contact with unexploited soils with adequate
Pi levels.
Therefore, the relationship between organ growth and Pi
starvation responses is complex and involves feedback
control. Root growth is required for the ability to acquire Pi
from new sources, but, simultaneously, root growth is likely
to modify the level of demand for Pi. Thus, the Pi starvation
state probably serves to extend the plant’s ability to sustain
root growth for the longest possible time, although this has
not yet been experimentally demonstrated. The degree and
duration of root growth sustenance has implications for
fitness, and Arabidopsis accessions from different environments have distinct efficiencies for phosphate use (Narang
et al., 2000).
The recursive nature of the relationship between phosphate demand and organ growth, where the levels of organ
growth feed back on the magnitude of Pi demand, has
important implications. We propose that other factors, e.g.
plant growth regulators or environmental cues, that affect
plant growth behaviour, will also affect Pi starvation
responses by affecting the magnitude of Pi demand. For
example, it has been suggested that cytokinins (FrancoZorrilla et al., 2002; Martin et al., 2000) suppress Pi starvation responses. These reports raise the question as to
whether the effects of these growth regulators on PSR gene
expression are direct or mediated by the known inhibitory
effects of cytokinins on growth.
Here, we examine the relationship between growth and Pi
starvation responses. We show that the magnitude of Pi
starvation-responsive gene expression is sensitive to the
balance between Pi supply and Pi demand. Media supplements or treatments that promote growth under Pi starvation conditions exacerbate Pi starvation responses, while
those that diminish growth repress Pi starvation responses.
The treatments that affect growth also affect Pi levels in
shoot and root organs. Evidence is provided that the
magnitude of cell division activity determines the demand
for Pi in Pi-starved plants, and provides a crucial cue for
phosphate starvation signalling pathways.
Results
Pi starvation reduces the root apical growth rate by inhibition of cell expansion
To examine the relationship between phosphate starvation
responses and plant growth, we first analysed the root
growth of Arabidopsis seedlings grown on media with different Pi concentrations. From 4–5 days after germination
onwards, growth of the primary root became responsive to
external Pi (Figure 1a). Roots of seedlings grown on 2500 lM
Pi displayed the highest apical growth rate, and growth in
media with 125 lM Pi led to a reduced root growth rate
(Figure 1a). We examined the size of mature cortical cells to
determine whether these differences in organ growth were
due to differential cell expansion or altered rates of cell
proliferation in the meristem (Figure 1b). Cortical cells in
5-day-old plants grown under different conditions did not
significantly differ in size (mean length 141 2.8 lM), but, at
11 days, their length in roots of plants grown in 125 lM Pi
was approximately 60% of that in roots from plants grown in
2500 lM Pi (Figure 1b), consistent with observations made
by Williamson et al. (2001).
Growth history, Pi supply and molecular responses to
phosphate starvation
We reasoned that prior growth of Arabidopsis seedlings on
media with high as opposed to low-Pi supplements, while
not strongly affecting proliferation, would affect their ability
to accumulate Pi, and hence their subsequent response to Pi
starvation. Plants pre-grown in 2500 lM Pi accumulated 34
and 14.5 nmol Pi mg)1 fresh weight (FW), respectively, in
shoot and root tissues. In contrast, plants pre-grown in
125 lM Pi had a lower Pi content and contained 8.5 and
6.9 nmol Pi mg)1 FW, respectively, in shoot and root tissues.
We then analysed the effect of such different levels of Pi
supply on PSR gene induction in plants transferred to a
Pi-depleted medium (Figure 1c). We used molecular markers
representing different pathways involved in Pi-starvation
responses: the high-affinity phosphate transporter PHT1;1
(At5g43350), the monogalactosyl-diacylglycerol synthase
MGD3 (At2g11810) involved in galactolipid synthesis, the
UDP-sulfoquinovose synthase SQD1 (At4g33030) involved
in sulfo-lipid synthesis, the purple acid phosphatase ACP5
(At3g17790), and the putative ribo-regulator At4 (At5g03545)
(data for ACP5 and At4 not shown). These PSR markers are
induced by phosphate starvation in Arabidopsis (Hammond
et al., 2003; Misson et al., 2005; Wu et al., 2003).
Expression of PSR genes, particularly of MGD3 and SQD1,
was quickly induced after plants were transferred from
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Growth modulates Arabidopsis phosphate starvation responses 547
(a)
(b)
(c)
observed in plants pre-grown in 125 lM Pi. In contrast, PHT1;1
and MGD3 expression from the same plants had only been
induced to 10–20% of the levels observed in plants pre-grown
in 125 lM Pi. These differences in the timing and magnitude
of PSR gene induction inversely correlated with the supply of
Pi accumulated in plants during pre-growth. The delayed
onset of PSR gene expression in plants pre-grown under high
as opposed to low-Pi conditions (Figure 1c) suggests that
these starvation responses are triggered by internal cues, and
not by the external Pi concentration, in agreement with a
previous report examining tomato cell cultures (Köck et al.,
1998). Together, these results indicate that pre-growth in
media with varying Pi levels led to proportionally different
levels of Pi supply, which strongly influenced the magnitude
of Pi starvation responses when these plants were transferred
to Pi-depleted media.
Enhanced proliferation increases phosphate starvation responses
Figure 1. Root apical growth and gene expression responses to different Pi
availability.
(a) Reduction of primary root apical growth in Pi-limiting conditions. Root
length was scored in Arabidopsis seedlings grown on vertical plates in halfstrength MS with either 125 or 2500 lM phosphate added. Error bars show
SEM.
(b) Inhibition of cell expansion in low-Pi media. The cell length of fully
expanded cortical cells was measured at 11 days. Error bars show SEM.
(c) Timing of onset and magnitude of phosphate starvation-responsive gene
expression. To obtain adequate amounts of RNA, these experiments were
performed with seedlings grown in flasks. After pre-growth in replete
(2500 lM Pi) or limiting (125 lM Pi) media, these were exchanged for media
completely lacking Pi, or replaced with the same media. Roots were harvested
at the time points indicated, and RNA levels of PHT1;1, MGD3 and SQD1 were
determined using quantitative RT-PCR. For clarity of presentation, data from
plants grown in the presence of 125 or 2500 lM Pi, in which PHT1;1, MGD3 and
SQD1 RNA were not induced, are not shown. Error bars show SEM.
medium with 125 lM Pi to a medium without added Pi
(Figure 1c). However, gene induction was markedly delayed
and reduced in plants pre-grown on medium with 2500 lM Pi
(Figure 1c). In these experiments, SQD1 expression appeared
most responsive to changes in Pi availability: After approximately 3 days growth in Pi-depleted medium, plants pregrown in medium supplemented with 2500 lM Pi had already
induced SQD1 expression to approximately 50% of that
Our data raised the possibility that the balance of Pi supply
(reserves accumulated during growth in Pi-replete conditions) and demand (magnitude of growth activity) controls
the level of PSR gene induction. To test this hypothesis,
experiments were designed to affect demand by manipulating shoot and root organ growth rates through altered
nutrient availability, treatments with growth regulators or
abiotic stress. In these experiments, we focused on MGD3 as
a representative molecular marker for phosphate starvation
responses because of its large response amplitude, but
PHT1;1 and At4 gave qualitatively very similar results.
To stimulate growth, we increased the levels of sugar
supplemented to the media. Seedlings grown hydroponically in Pi-replete media for 12 days were then shifted to
Pi-depleted medium, supplemented with either 0.3% or 2%
sucrose (Suc), glucose (Glu) or 3-o-methyl glucose (3-omGlu), respectively. 3-o-mGlu, a metabolically inert (Cortes
et al., 2003) glucose analogue, was used as an osmotic
control in these experiments. The addition of 2% Suc or Glu
to Pi-depleted media specifically promoted root growth,
while 2% 3-o-mGlu was growth-inhibitory (Figure 2a), possibly due to its ability to compete with hexoses for uptake by
monosaccharide transporters (Cortes et al., 2003). Root
growth in Pi-depleted media with 2% Suc more than doubled
after 6 days of phosphate starvation when compared with
controls (Figure 2a), while shoot growth was reduced to
approximately 60% of controls (not shown).
We analysed cortical cell length to examine whether root
growth stimulation under these conditions was caused by
increased proliferation or cell expansion. In Pi-replete media,
cortical cell length was 209.5 4.9 lM (n = 89) in low-Suc
medium and 214.1 5 lM (n = 89) in high-Suc medium,
while in Pi-depleted media, cortical cell length was
87.2 3.1 lM (n = 89) in low-Suc medium and
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548 Fan Lai et al.
81.6 2.1 lM (n = 88) in high-Suc medium. These data
indicate that increased root growth at high-sugar concentrations is caused by increased cell production.
Most PSR genes, including MGD3 (Awai et al., 2001), are
predominantly expressed in roots; we therefore initially
Roots mass (FW) (g)
(a)
(b)
focused our gene induction analysis in these experiments on
this organ. The addition of 2% Suc or Glu to the medium, but
not of 3-o-mGlu, strongly enhanced MGD3 expression levels
in response to Pi starvation in roots when compared with
control medium (Figure 2b), indicating that only the sugar
supplements that stimulated growth affected PSR gene
expression levels under Pi-depleted conditions.
We next examined whether elevated PSR gene expression
in plants supplemented with 2% Suc or Glu correlated with
altered Pi homeostasis. During the time course of the
experiment, phosphate levels in roots declined from an
initial concentration of approximately 15 to approximately
3–5 nmol Pi mg)1 FW (Figure 2c). The initial rate of decline in
Pi concentration 2 days after transfer to Pi-depleted media
was modestly, but reproducibly, higher in plants grown in
the presence of 2% Suc or Glu, when compared with
controls (Figure 2c). At later time points, root Pi concentrations in plants grown in the presence of 2% Suc or Glu were
not significantly different from those of controls (Figure 2c).
However, root Pi levels did not decline strongly when plants
were grown in the presence of 3-o-mGlu (Figure 2c),
consistent with the absence of PSR gene induction (Figure 2b).
Taken together, these data suggested that sugar supplements to Pi-depleted media transiently increased the
demand for Pi and durably enhanced PSR gene expression.
Elevated PSR gene expression and increased demand for Pi
correlated with enhanced root growth. Conversely, root
growth inhibition durably decreased the demand for Pi and
suppressed PSR gene induction. We next performed experiments to dissect the relationship between organ growth and
Pi starvation responses.
Cytokinin affects phosphate starvation responses due to its
effects on growth
It has been proposed that PSR genes are suppressed by
cytokinins (Franco-Zorrilla et al., 2002; Martin et al., 2000).
To test whether this control is direct, or, alternatively, whether it is a consequence of the effects of cytokinins on
growth, we examined growth, PSR gene expression
Phosphate in roots (nmol mg–1 FW)
(c)
Time after transfer to new media (days)
Figure 2. Elevated growth enhances phosphate starvation responses.
(a) Media supplemented with metabolically active sugars [sucrose (Suc),
glucose (Glu)] but not the inactive sugar 3-o-methyl glucose (3-o-mG)
promotes root growth. Arabidopsis seedlings were pre-grown in replete
(1250 lM Pi) media, then shifted to media without added Pi, with or without
sugar supplements. The fresh weight (FW) of the root system of five plants per
flask was measured every 2 days; error bars show SEM.
(b) Pi starvation-induced MGD3 expression is enhanced only by metabolically
active sugars. Arabidopsis seedlings were grown hydroponically with or
without a supplement of 2% Suc, Glu or 3-o-mG. MGD3 gene expression in
samples from the experiment shown in (a) was monitored by quantitative RTPCR; error bars show SEM. Note the break in the y axis.
(c) Root organ Pi content is inversely proportional to growth activity.
Phosphate content was measured in aliquots of samples from the experiment
shown in (a). Error bars show SEM.
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Growth modulates Arabidopsis phosphate starvation responses 549
Figure 3. Cytokinin affects growth and phosphate starvation responses in
shoots and roots in opposite ways. All data are from the same representative
experiment. Seedlings were pre-grown in Pi-replete (1250 lM Pi) half-strength
MS medium in flasks, shifted to Pi-depleted media with or without 10 or
2.5 lM kinetin, and grown for 6 days. Samples were taken at the time points
indicated.
(a) Kinetin (10 lM) promotes shoot growth and inhibits root growth. At the
indicated times, flasks were harvested and fresh weight (FW) determined.
Error bars show SEM.
(b) Phosphate starvation responses are enhanced in shoots treated with
kinetin in a dose-dependent manner. RNA was isolated from shoot tissues of
plants grown in the presence of either 10 or 2.5 lM kinetin, and MGD3 gene
expression was assessed by quantitative RT-PCR. Error bars show SEM.
(c) Phosphate starvation responses are inhibited in a dose-dependent manner
in roots treated with kinetin. RNA was isolated from root tissues, and MGD3
gene expression was determined by quantitative RT-PCR. Error bars show
SEM.
(d) Organ-specific phosphate content changes are inversely proportional to
growth activity. Plants were either not treated or treated with 10 lM kinetin.
Organ samples were extracted and soluble phosphate content was determined by colorimetric assay. Error bars show SEM.
expected to change in inverse proportion to organ-specific
growth activity. In kinetin-treated shoots, which were producing more biomass, steady-state Pi levels were 40–50%
Organ mass (FW) (g)
(a)
(b)
(c)
(d)
Phosphate in organs (nmol mg–1 FW)
responses and Pi concentration in Pi-depleted media after
kinetin treatments. If PSR gene induction were directly
suppressed by cytokinin, we would expect to observe a
reduction in Pi starvation-induced gene expression in all
organs. However, cytokinins affect plant organ growth by
enhancing the activity of shoot, while reducing the activity of
root meristems (Skoog and Miller, 1957; Werner et al., 2001).
Therefore, if the magnitude of PSR gene induction were
determined by organ growth activity, we would expect to
observe opposite effects in root and shoot organs of cytokinin-treated plants.
Plants were grown hydroponically and shifted to a
Pi-depleted medium, with or without kinetin supplements.
As expected, the addition of kinetin to the media stimulated
shoot growth and reduced root growth: shoot growth was
increased by approximately 30% and root growth was
reduced to approximately 55% of that of controls in the
presence of 10 lM kinetin (Figure 3a). Next, we examined
organ-specific PSR gene expression in these plants (Figure 3b,c). In shoots, kinetin treatments strongly enhanced
MGD3 gene expression in a dose-dependent manner (Figure 3b). Conversely, in roots, kinetin treatments strongly
suppressed MGD3 gene expression in a dose-dependent
manner (Figure 3c). Suppression was also observed without
kinetin treatments in wild-type plants when compared with
mutant backgrounds with reduced cytokinin sensitivity: the
expression of MGD3 was always higher in cre1-2 and cre1-4
mutant plants grown in Pi-depleted media, when compared
with the wild-type (Figure S1). Together, these results
support the notion that kinetin acts to modulate the level
of growth activity, which in turn determines the magnitude
of PSR gene expression.
We next examined whether organ-specific Pi homeostasis
followed a similar pattern. We reasoned that if kinetin
exerted its effects by affecting meristem activity, and thus
shoot or root organ-specific Pi demand, Pi levels would be
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550 Fan Lai et al.
lower than in controls (Figure 3d). In contrast, Pi levels in
kinetin-treated and hence growth-inhibited roots, were
around twice those observed in controls (Figure 3d).
We concluded that elevated organ growth correlated with
lower Pi levels and higher PSR gene expression responses,
and vice versa. Therefore, our observations supported the
notion that kinetin acts through the control of meristem
activity and organ growth to affect the magnitude of PSR
gene expression. However, we cannot rule out the possibility that kinetin inhibits Pi translocation from the root to the
shoot.
induction in Pi-depleted conditions was reduced to 10–20%
of the levels observed in Pi-depleted half-strength Johnson
medium (Figure 4a). Root growth was inhibited when Arabidopsis seedlings were exposed to 1% mannitol or 18 mM
total N (15 mM nitrate and 3 mM ammonium) (Figure 4b,c).
Root growth inhibition was observed for the total root
(a)
Osmotic conditions that inhibit growth also reduce Pi
starvation responses
To further strengthen the hypothesis that the demand for Pi,
specified by the scale of growth activity, determines the
magnitude of PSR gene expression, we examined the effects
of other supplements known to affect root growth. We tested
the effects of osmotic stress-inducing levels of mannitol, KCl
or nitrogen (N) supplements on root growth and MGD3
expression levels (Figure 4). High-osmolarity environments
inhibit root and plant growth (Hasegawa et al., 2000).
These experiments were conducted in half-strength Johnson medium (Johnson et al., 1957). Johnson medium is low
in solutes, which makes it much easier to discern plant
responses to osmotic stress or high-nitrogen supplements.
Pi-depleted half-strength Johnson medium contains 8 mM
total nitrogen (N) and 1 mM KCl. To increase osmolarity, we
supplemented the medium with either 1% mannitol, 10 mM
total N or 19 mM KCl. Under these conditions, PSR gene
Figure 4. Osmotic stress suppresses phosphate starvation responses, reduces root growth, and inhibits cell division. Seedlings were grown on plates or
hydroponically in the presence of phosphate for 11 days, and then transferred
to Pi-depleted media (8 mM total N) with or without the supplements indicated
(N to 18 mM, KCl to 20 mM or mannitol to 1% final concentration, respectively)
or to Pi-replete half-strength Johnson medium. Plants were sampled every
2 days.
(a) Seedlings were harvested at the indicated time points, RNA extracted and
steady-state MGD3 expression was monitored by quantitative RT-PCR; error
bars show SEM. Note the break in the y axis.
(b) Root length was measured in plants grown on vertically oriented Petri
dishes after transfer to media without added Pi supplemented with either
19 mM KCl or 1% mannitol or unsupplemented; error bars show SEM, and are
visible when larger than the symbol. Note the break in the x axis.
(c) Root fresh weight was determined for plants grown in Pi-depleted low-N
medium (8 mM total N) or high-N medium (18 mM total N). Error bars show
SEM.
(d, e) Nine-day-old seedlings were transferred to Pi-depleted media with or
without the supplements indicated or to Pi-replete medium. Plants were
analysed after 6 days. Error bars show SEM. Different letters on the columns
indicate that values are significantly different (P < 0.01). (d) Elevated
osmolarity of the medium reduces cell division frequency in roots. Plants
transformed with a cyclin CYCB1;1–GUS fusion (FA4C; Colon-Carmona et al.,
1999) were histochemically analysed for mitotic activity and the frequency of
mitotic cells determined per root tip. Note the break in the y axis.
(e) KCl enhances cell expansion. Cortical cell size was measured on fully
expanded cells in the interval between 0.5 and 1 cm from the apex of mounted
roots.
(b)
(c)
(d)
(e)
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Growth modulates Arabidopsis phosphate starvation responses 551
system (Figure 4c) as well as for primary root apical growth
(Figure S2) when grown in 18 mM total N. N is more limiting
for shoot growth than for root growth (Marschner, 1995;
Thornley, 1972); therefore, as expected, we observed that
18 mM N promoted shoot growth and Pi consumption, while
the opposite was true for roots (Figure S3).
Interestingly, root growth was slightly stimulated when
half-strength Johnson medium was supplemented with
19 mM KCl (Figure 4b,c). To resolve whether KCl possibly
had a unique role that uncoupled the tight correlation we
had observed between plant organ growth, phosphate
demand and the magnitude of phosphate starvation responses in all previous experiments, we examined the
growth response in these roots in more detail. Organ growth
is the aggregate result of cell growth and cell division in
meristems, and post-mitotic cell expansion. As potassium is
crucial for post-mitotic cell expansion, we examined cell
division and expansion with the aim of identifying which of
these processes cued Pi demand.
Mitotic activity and cell size were examined in the roots of
plants grown with different supplements (Figure 4d,e).
Cortical cells in Pi-depleted roots grown in the presence of
20 mM KCl were approximately 35% longer (Figure 4e),
while root length in the same growth interval (6–8 days,
Figure 4b) was only 4% greater, when compared with nonsupplemented Pi-depleted medium. This indicated that,
while 20 mM KCl inhibited cell division, 1 mM KCl was
limiting for cell expansion in half-strength Johnson medium. On balance, despite inhibition of proliferation, root
growth was slightly increased in 20 mM KCl, mediated by
increased cell expansion. By contrast, 1% mannitol supplemented to Pi-depleted media did not significantly change
mature cortical cell size (Figure 4e). To confirm that cell
division activity in roots was impaired under all conditions
that diminished PSR gene induction, we measured the
mitotic activity in roots. The addition of mannitol, KCl or
kinetin reduced cell division to approximately 70%, 65% and
60%, respectively, of the mitotic activity observed in roots
grown in Pi-depleted media (Figure 4d, Figure S4). The
effect of these treatments on cell division was rapid and
was already clearly evident after 12 h (Figure S4).
Cell division activity specifies Pi demand in Pi-starved
conditions.
These observations raised the possibility that proliferation in
meristems specifies the magnitude of demand for phosphate and of PSR gene induction. To examine this hypothesis, we treated plants growing in Pi-depleted medium
with colchicine, which interferes with the formation of a
functional mitotic spindle and therefore blocks cells in
mitosis. After 24 h, we did not observe any cells in telophase
(n = 628), indicating that, as expected, cells were not passing
through anaphase. While PSR gene induction continued to
increase in Pi-depleted plants, PSR gene expression was
strongly suppressed in Pi-depleted plants treated with colchicine (Figure 5a). We next examined whether the suppression of PSR gene induction was a general feature of cellcycle-arrested cells. Plants growing in Pi-depleted media
were treated with propyzamide and colchicine for 24 h.
Propyzamide reversibly binds b-tubulin and prevents
microtubule polymerization, thus interfering with the formation of a functional mitotic spindle. As expected, inhibition of cell division suppressed PSR gene expression
(Figure 5b). The inhibitors were then washed out and we
monitored PSR gene expression. Suppression of PSR gene
expression by inhibitors of division was reversible, and at
the end of the time course plants responded to Pi treatments
(Figure 5b).
Proliferation in meristems depends on prior growth of
cells. Colchicine blocks cells in mitosis without inhibiting cell
growth (Foard et al., 1965), allowing us to distinguish whether cell growth (and pre-requisite metabolism) or cell
division cue Pi starvation responses. Despite the mitotic
arrest (Figure 5a), cells in the meristem continue to grow in
the presence of colchicine: in Pi-depleted medium, epidermal
cells in trichoblast positions were 5.47 0.11 lM long
(n = 158), while those treated with colchicine were
11.68 0.19 lM long (n = 84). We conclude that cell-cycle
activity specifies the demand for Pi, providing a cue to
quantitatively modify the magnitude of PSR gene expression.
Discussion
Analysis of Pi-starvation responsive (PSR) gene expression
in plants with distinct levels of Pi supplies due to pre-growth
in media with different levels of Pi availability revealed that
the magnitude of their induction after transfer to a Pidepleted environment is sensitive to the balance of Pi supply
and demand. We found that growth-promoting conditions
enhanced Pi starvation responses and accelerate depletion
of available Pi, while growth-inhibitory conditions repressed
Pi starvation responses and retard depletion of available Pi,
by subjecting plants to different growth conditions after
transfer to Pi-depleted media. By selective inhibition of cell
expansion (by limiting KCl) or cell division (by inhibitors of
cytokinesis), we show that cell-cycle activity determines the
demand for Pi, and therefore, under conditions where supply
is limiting, the magnitude of PSR gene expression.
Economic notions are useful to conceptualize phosphate
pool dynamics
PSR genes are not immediately induced in roots of Arabidopsis plants transferred to Pi-depleted conditions (Figure 1c), indicating that these responses are not cued by the
Pi available in the environment. The timing and magnitude
of PSR gene induction suggests that it is sensitive to the life
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552 Fan Lai et al.
history of a plant. Growth in Pi-replete conditions (defined as
above the threshold that would activate transition towards a
phosphate starvation state involving concerted changes to
growth behaviour, metabolism and nutrient transport) generates a supply in excess of ongoing demand, which is
deposited either directly in the vacuoles of leaf organs
(Mimura, 1999) or as readily mobilized forms such as in
membrane phospholipids (Cruz-Ramı́rez et al., 2006;
Dörmann and Benning, 2002). Once plants experience a Pidepleted environment, demand exceeds the supply available in the freely convertible pool of phosphate (e.g. Pi, ATP),
necessitating a shift towards a new homeostatic state, the Pi
starvation state. A hallmark of the transition towards this
state is the mobilization of reserves (preferentially from the
supplies stored in shoot tissues), and net Pi transport to
roots (Dong et al., 1998). In roots, this supply would be
incorporated into the freely convertible pool to sustain
metabolism and to satisfy the demand for organ growth
required to contact new sources of phosphate. However, a
fraction of this pool is continuously withdrawn into an irretrievable Pi pool, when cells synthesize DNA and residual
irreplaceable phospholipids, thereby exacerbating Pi starvation responses over time. Therefore, the notions of Pi
supply and demand are useful to conceptualize the dynamic
changes to the phosphate budget of a plant in Pi starvation
conditions, because they incorporate the plant’s life history
and its current environment.
(a)
(b)
Cell division control as target of environmental and plant
growth regulator signalling pathways
In a Pi-depleted environment, conditions that enhance organ
growth (Figures 2a and 3a) also exacerbate PSR gene
expression (Figures 2b and 3b). In contrast, environments
that inhibit growth (Figures 2a, 3a and 4b,c) specifically reduce mitotic activity (Figure 4d, Figure S4) and reduce Pi
starvation responses (Figures 2b, 3c and 4a). Importantly,
these environments not only affect the magnitude of PSR
gene expression, but also result in altered levels of soluble Pi
(which reflects the freely inter-convertible phosphate pool):
When growth is inhibited, Pi content rises, while, when
growth is promoted, Pi levels initially decline more rapidly
relative to controls. Such comprehensive responses suggest
that sucrose, kinetin and osmotic stress-inducing supplements in a Pi-depleted environment do not directly target
PSR gene expression control. Instead, our results indicate
that they target the more basic plant growth control
machinery in meristems to modulate cell division activity
(Figure 4d). This conclusion is supported by the observation
that inhibition of cell division activity completely suppresses
PSR gene induction in Pi-depleted conditions (Figure 5a,b).
We propose that, in Pi-depleted conditions, the magnitude of
PSR gene induction reflects the level of Pi demand generated
by cell production.
Figure 5. The level of cell division activity specifies the magnitude of
phosphate demand. Seedlings were grown in the presence of phosphate for
9 days and then transferred to Pi-depleted media for 5 days. Cell-cycle
inhibitors or sham were added at t = 0. Plants were analysed after 24 h (a) or
in a time-course experiment (b).
(a) PSR gene induction is repressed when cell division is inhibited. Cell
division was inhibited by arresting cells in M phase by addition of colchicine
to a final concentration of 0.05%. Plants were harvested after 24 h, RNA
extracted and steady-state MGD3 expression was monitored by quantitative
RT-PCR; error bars show SEM.
(b) The effects of cell division inhibitors on PSR gene expression are
reversible. Plants were treated with propyzamide (12 lM) or colchicine
(0.01%) for 24 h. Inhibitors were removed by three washes in Pi-depleted
medium, and plants were incubated for 24 h. At t = 48 h, Pi (2500 lM) was
added to the media and plants were sampled after 12 h. Plants were harvested
at the time points indicated, RNA extracted and steady-state MGD3 expression monitored by quantitative RT-PCR; error bars show SEM.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 545–556
Growth modulates Arabidopsis phosphate starvation responses 553
How is the level of Pi demand monitored in cells?
Phosphate has a dual nature: it functions as a nutrient,
materially required for biosynthesis, and as a signal that is
perceived by cellular signalling pathways. It is perception of a
‘low-Pi’ cue that triggers the transition to the phosphate
starvation state, much preceding Pi consumption (Figures
2b,c and 3b–d). This can be demonstrated by use of
phosphonate, a structural analogue of Pi, to mimic Pi
perception by plant cells: phosphonate suppresses the
onset of PSR gene expression when plants are transferred
to Pi-depleted conditions (Ticconi et al., 2001), and, in
Pi-depleted plants, phosphonate treatments are sufficient to
down-regulate Pi starvation-induced high-level PSR gene
induction (Lai and Doerner, unpublished results). Phosphonate is thought to not participate in metabolism, and function
exclusively as signalling molecule (Abel et al., 2002),
although, for long-term treatments, this remains to be
experimentally verified. Interestingly, after plants are transferred to Pi-depleted conditions, phosphonate can sustain
root growth and cell division (Ticconi et al., 2004), while
suppressing PSR gene induction. This seemingly contradicts
our tenet that cell-cycle activity specifies the magnitude of
PSR gene expression, because cell proliferation should
generate a demand for Pi that cannot be satisfied in
Pi-depleted conditions and hence lead to PSR gene induction.
However, high-concentrations of phosphonate are perceived
as ‘high-phosphate’ by the plant, and hence such plants are
subjectively not Pi-starved. This deceit can be sustained for
some time in the continued presence of phosphonate, as
each replication cycle of the diploid genome withdraws only a
small fraction of the Pi from the total cellular phosphate pool.
The fundamental response of plants to environmental
change is altered growth behaviour, e.g. changes to organ
growth rate or pattern, or tropic responses. We show here
that Pi limitation reduces cell proliferation (Figure 4d). In cell
cultures, progression from G1 to S phase is sensitive to Pi
availability (Amino et al., 1983; Sano et al., 1999), and it is
likely that this holds true for Arabidopsis. We also show that
inhibition of cell division reduces Pi demand, and hence PSR
gene induction. This implies the existence of a feedback loop
that couples proliferation with Pi signalling mechanisms.
Work is in progress to identify the mechanisms by which
progression through the cell division cycle is made sensitive
to Pi availability, and how cell-cycle activity cues Pi sensing
and signalling.
Other environmental cues similarly target the meristem,
specifically cell division activity. For example, elevated ABA
levels due to reduced water potential, or salt stress, induce
KRP1, which interacts with CDKA1 and CYCD3;1 to inhibit
entry into S phase (Wang et al., 1998). It has previously been
suggested that cytokinins repress PSR gene expression
(Franco-Zorrilla et al., 2002; Martin et al., 2000). Recently,
sugars (Franco-Zorrilla et al., 2005), auxin (Kobayashi et al.,
2006) and ABA (Shin et al., 2006) were added to the list of
cues positively or negatively affecting PSR gene expression.
It is notable that these cues all affect cell proliferation.
These findings have led to suggestions of complex
models involving ‘multi-dimensional cross-talk’ between
numerous signalling pathways (Franco-Zorrilla et al., 2005).
However, our data reveal that these models do not
adequately consider the effects of cytokinins, sugars, etc.
on growth and phosphate levels. By considering growth as
the central response by plants to environmental change, we
propose a more parsimonious model in which conditions
that promote organ growth by proliferation enhance Pi
starvation responses (due to increased demand), whilst
those inhibiting cell production reduce the magnitude of Pi
starvation responses (due to diminished demand).
At the organ level, longitudinal growth is largely driven by
increased turgor pressure associated with growth of the
vacuole against a background of cell-wall loosening.
Increasing cell volume by cell expansion does not involve
commensurate increases in cellular protein content. This
raises the question of why cell expansion is inhibited in Pi
starvation, as this could be considered to be a more frugal
use of limiting Pi. We speculate that this is not the case
because cell expansion is associated with endoreplication,
which on a per cell basis is liable to consume more Pi
than the production of smaller, but diploid cells. It is
experimentally testable whether endoreplication is reduced
in Pi-depleted conditions.
Growth control pathways
Our growth data suggests that carbon (Figure 2) and nitrogen
(Figure S3) can still limit plant growth, even when Pi is limiting. This observation contrasts with the ‘law of the minimum’ originally proposed by Sprengler in the 19th century
and later championed by Liebig (van der Ploeg et al., 1999),
which posits that any nutrient, when limiting, would control
plant growth, regardless of the availability of other nutrients.
Modelling of metabolic control of plant growth (Bloom et al.,
1985; Thornley, 1972) has suggested that C and N metabolism
provide the primary and dominating cues for growth control
pathways. Our results experimentally support the notion that
C and N metabolism provide a cue that over-rides Pi signals
for growth control pathways, although we presently cannot
rule out the possibility that 2% sugar interferes with phosphate sensing. This suggests that in plants, just as in yeast,
insect and mammalian cells, the availability of carbon and
nitrogen are primary inputs for growth control.
It will be interesting to determine which signalling
pathways primarily control cell proliferation rates under
Pi-depleted conditions. In the eukaryotic model systems
Saccharomyces cerevisiae, Drosophila and humans, the
respective orthologues of the TARGET OF RAPAMYCIN
(TOR) protein kinase have been shown to be at the top of a
signalling hierarchy that ultimately controls cell proliferation
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 545–556
554 Fan Lai et al.
(Hay and Sonenberg, 2004; Schmelzle and Hall, 2000).
Arabidopsis has a TOR orthologue; however, this has not
yet been functionally characterized in great detail (Menand
et al., 2002, 2004). DELLA genes also regulate plant growth
by mediating gibberellic acid-dependent signals, and specifically serve to restrain growth in adverse conditions
(Achard et al., 2006). Experiments are in progress to determine the role of these pathways in controlling plant growth
in response to altered nutrient availability.
Experimental procedures
Plant growth and media
All plants were grown either in half-strength MS medium (Murashige and Skoog, 1962) or in half-strength Johnson medium
(Johnson et al., 1957) with the amounts of phosphate indicated.
Arabidopsis (Col-0) seeds were surface-sterilized in diluted bleach.
Seeds were re-suspended in 0.1% micro-agar (Duchefa, www.
duchefa.com) and stratified at 4C for 2 days. For root growth
experiments, seeds were plated on medium solidified with 1%
micro-agar (Duchefa) on vertically oriented square plates, and
grown at 21C with a 16 h day/8 h nigh photoperiod. Root growth
was marked every 12 or 24 h. For analysis of gene expression, five
seedlings were grown in conical flasks containing 5 ml media and
placed on a shaker (40 rpm). To induce starvation, roots were first
rinsed with 5 ml 0.2 mM CaCl2 (in 5 mM MES, pH 5.5) to complex
residual phosphate and then transferred to medium lacking Pi.
Phosphate measurements
The ascorbate–molybdate method of phosphate determination
(Murphy and Riley, 1962) was adapted to determine Pi levels in
tissues and media. After FW determination, tissues were homogenized in sterile H2O using glass beads in a bead mill and centrifuged (20 K · g). Supernatants were stored at )20C until use. All
samples were analysed at least in duplicate, and errors are stated as
standard error of the mean (SEM).
RNA isolation and quantitative RT-PCR
Tissue samples were frozen in liquid nitrogen at harvest and
homogenized in TRIzol reagent (Invitrogen; http://www.invitrogen.
com/). RNA pellets were re-suspended, precipitated with 2 M
LiCl, and re-suspended in DEPC-H2O. RNA (0.5 lg) was reversetranscribed with Superscript reverse transcriptase (Invitrogen),
using oligo(dT) primers or gene-specific primers. PCR reaction
components included 3 ll diluted cDNA sample, 10 ll 2 · QPCR
Master Mix, containing 0.025 units ll)1 Thermo-Start enzyme
(Abgene, www.abgene.com) 4.2 ll sterile H2O, 2 ll SYBR Green
(1:10 000 dilution of 10 000 · concentrate in DMSO, Molecular
Probes, http://probes.invitrogen.com), 0.4 ll (100 ng ll)1) forward
primer and 0.4 ll (100 ng ll)1) reverse primer. The following primer
sets were used: eIF4A (At3g13920) (cDNA), 5¢-TTCGCTCTTCT
CTTTGCTCTC-3¢, 5¢-GAACTCATCTTGTCCCTCAAGTA-3¢; eIF4A (genomic), 5¢-CATTTTCTCCGCACATCATC-3¢, 5¢-AAACTGTGTGCCTT
CTGGTG-3¢; At4 (At5g03545), 5¢-GGATGGCCCCAAACACAAG-3¢, 5¢TAAACCGGAAACAAAGTAAACACG-3¢; MGD3 (At2g11810), 5¢-TG
CCACCGTACATGGTTC-3¢, 5¢-TTGTCCTATTGGATTACTTTCTTTA
GAG-3¢; SQD1 (At4g33030), 5¢-CTTAGCAAAGTTCATGATTCG
CAC-3¢, 5¢-GCCTCTCGTCTGACCACCTTTAC-3¢; PHT1;1 (At5g43350),
5¢-CTGCCAAGCTGATTAAGAGC-3¢, 5¢-GACAGAGCACAAGATCAT
CATTAC-3¢; ACP5 (At3g17790), 5¢-CACGGCGAGTCTGAGTTTGCTG3¢, 5¢-CTCTCCAATTTTTCCCATCTGATAAGC-3¢. Each quantitative
PCR reaction was performed in quadruplicate in an iCycler iQ (BioRad; http://www.bio-rad.com/), using the 490 nm filter for SYBR
green fluorescence detection. Each cDNA sample was amplified
with eIF4A (cDNA) primers to assess total RNA content. Amplification with eIF4A (genomic DNA) primers was also performed to
assess genomic DNA contamination when carrying out PCR with
At4 primers. eIF4A encodes the constitutively expressed eukaryotic initiation factor 4A (Metz et al., 1992) that has been shown to
provide a good control for constitutive gene expression (Czechowski
et al., 2005). All other primer combinations contained at least
one primer designed to cross an intron, and thus only cDNA was
amplified.
For quantification of RT-PCR, threshold values for amplification
were kept constant for all samples. To normalize for cDNA loading,
threshold cycle differences were obtained by subtracting the mean
threshold cycle for each gene (a) from the mean threshold cycle of
eIF4A amplification from the same cDNA sample (b). Relative
amounts were then calculated by subtracting this value from the
mean threshold cycle of the sample designated to be the reference
(c). To represent relative amounts, these logarithms were inversed
using the equation: relative amount = 2c) (b ) a).
Cell measurements
The Arabidopsis line Q8, which expresses a GFP fusion to the plasma
membrane water channel protein PIP2A (Cutler et al., 2000), was
used to measure cell size (for experiments summarized in Figure 1)
after confirming that this line was indistinguishable from the wildtype in growth assays. Six roots from each growth condition and 10
cortical cells per root were imaged. Fluoview image analysis software (Olympus; http://www.olympus-global.com/) was used to calculate cortical cell length. In other experiments, cells were visualized
using DIC optics, photographed with a digital camera, and measured
using Image J. Mitotic frequencies were determined using the FA4C
background (Colon-Carmona et al., 1999, http://rsb.info.nih.gov/ij/).
The t test function implemented in MS Excel was used to test whether the observed differences between experimental conditions were
significant (P < 0.01). To assess chromosome morphology for the
colchicine experiment, roots were fixed with 3.7% formaldehyde,
rinsed in PBS and stained with DAPI for 1 h at a final concentration of
0.05 lg ml)1.
Acknowledgements
We thank the Samuel Roberts Noble Foundation, the Darwin Trust,
the Royal Society and the Biotechnology and Biological Sciences
Research Council (BBSRC) for support. We thank Audrey Wang,
Angie Ng Ah Sock and Melina Reisenberg for excellent assistance.
We thank Antonio Leyva for providing phr1-1, Tatsuo Kakimoto for
cre1-2 and cre1-4, and the Nottingham Arabidopsis Stock Centre
(NASC) for providing seed. We thank all members of the Doerner
and Ingram labs for discussions and anonymous reviewers for
valuable suggestions.
Supplementary material
The following supplementary material is available for this article
online:
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 545–556
Growth modulates Arabidopsis phosphate starvation responses 555
Figure S1. Elevated Pi-starvation responsive gene expression in
cytokinin-insensitive mutants.
Figure S2. High-concentrations of N (ammonium and nitrate) in the
media inhibit root apical growth.
Figure S3. Growth in high-N (nitrate and ammonium) inhibits root
growth and delays root Pi consumption while enhancing shoot
growth and accelerating shoot Pi decline in Pi-deplete media.
Figure S4. Rapid cell division responses to adverse growth conditions are observed in Pi-deplete media.
This material is available as part of the online article from http://
www.blackwell-synergy.com
References
Abel, S., Ticconi, C.A. and Delatorre, C.A. (2002) Phosphate sensing
in higher plants. Physiol. Plant., 115, 1–8.
Achard, P., Cheng, H., De Grauwe, L., Decat, J., Schoutteten, H.,
Moritz, T., Van Der Straeten, D., Peng, J. and Harberd, N.P. (2006)
Integration of plant responses to environmentally activated phytohormonal signals. Science, 311, 91–94.
Amino, S.-I., Fujimura, T. and Komamine, A. (1983) Synchrony induced by double phosphate starvation in a suspension culture of
Catharanthus roseus. Physiol. Plant., 59, 393–396.
Awai, K., Marechal, E., Block, M.A., Brun, D., Masuda, T., Shimada,
H., Takamiya, K., Ohta, H. and Joyard, J. (2001) Two types of
MGDG synthase genes, found widely in both 16:3 and 18:3 plants,
differentially mediate galactolipid syntheses in photosynthetic
and nonphotosynthetic tissues in Arabidopsis thaliana. Proc.
Natl Acad. Sci. USA, 98, 10960–10965.
Bates, T.R. and Lynch, J.P. (1996) Stimulation of root hair elongation
in Arabidopsis thaliana by low phosphorus availability. Plant Cell
Environ., 19, 529–538.
Bates, T. and Lynch, J. (2000a) The efficiency of Arabidopsis
thaliana root hairs in phosphate acquisition. Am. J. Bot., 87, 964–
970.
Bates, T. and Lynch, J. (2000b) Plant growth and phosphorus
accumulation of wild type and two root hair mutants of Arabidopsis thaliana (Brassicaceae). Am. J. Bot., 87, 958–963.
Bieleski, R. (1973) Phosphate pools, phosphate transport and
phosphate availability. Annu. Rev. Plant Physiol., 24, 225–252.
Bloom, A., Chapin, F. and Mooney, H. (1985) Resource limitation
in plants – an economic analogy. Annu. Rev. Ecol. Syst., 16, 363–
392.
Colon-Carmona, A., You, R., Haimovitch-Gal, T. and Doerner, P.
(1999) Spatio-temporal analysis of mitotic activity with a labile
cyclin–GUS fusion protein. Plant J., 20, 503–508.
Cortes, S., Gromova, M., Evrard, A., Roby, C., Heyraud, A., Rolin,
D.B., Raymond, P. and Brouquisse, R.M. (2003) In plants, 3-omethylglucose is phosphorylated by hexokinase but not perceived as a sugar. Plant Physiol., 131, 824–837.
Cruz-Ramı́rez, A., Oropeza-Aburto, A., Razo-Hernández, F.,
Ramı́rez-Chávez, E. and Herrera-Estrella, L. (2006) Phospholipase
DZ2 plays an important role in extraplastidic galactolipid biosynthesis and phosphate recycling in Arabidopsis roots. Proc.
Natl Acad. Sci. USA, 103, 6765–6770.
Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K. and Scheible,
W.-R. (2005) Genome-wide identification and testing of superior
reference genes for transcript normalization in Arabidopsis. Plant
Physiol., 139, 5–17.
Dong, B., Rengel, Z. and Delhaize, E. (1998) Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of
Arabidopsis thaliana. Planta, 205, 251–256.
Dörmann, P. and Benning, C. (2002) Galactolipids rule in seed
plants. Trends Plant Sci., 7, 112–118.
Foard, D.E., Haber, A.H. and Fishman, T.N. (1965) Initiation of lateral
root primordia without completion of mitosis and without cytokinesis in uniseriate pericycle. Am. J. Bot., 52, 580–590.
Franco-Zorrilla, J.M., Martin, A.C., Solano, R., Rubio, V., Leyva, A.
and Paz-Ares, J. (2002) Mutations at CRE1 impair cytokinin-induced repression of phosphate starvation responses in Arabidopsis. Plant J., 32, 353–360.
Franco-Zorrilla, J.M., Martin, A.C., Leyva, A. and Paz-Ares, J. (2005)
Interaction between phosphate-starvation, sugar, and cytokinin
signaling in Arabidopsis and the roles of cytokinin receptors
CRE1/AHK4 and AHK3. Plant Physiol., 138, 847–857.
Hammond, J.P., Bennett, M.J., Bowen, H.C., Broadley, M.R.,
Eastwood, D.C., May, S.T., Rahn, C., Swarup, R., Woolaway, K.E.
and White, P.J. (2003) Changes in gene expression in Arabidopsis
shoots during phosphate starvation and the potential for developing smart plants. Plant Physiol., 132, 578–596.
Hartel, H. and Benning, C. (2000) Can digalactosyldiacylglycerol
substitute for phosphatidylcholine upon phosphate deprivation
in leaves and roots of Arabidopsis? Biochem. Soc. Trans., 28, 729–
732.
Hartel, H., Dormann, P. and Benning, C. (2000) DGD1-independent
biosynthesis of extraplastidic galactolipids after phosphate
deprivation in Arabidopsis. Proc. Natl Acad. Sci. USA, 97, 10649–
10654.
Hasegawa, P.M., Bressan, R.A., Zhu, J.K. and Bohnert, H.J. (2000)
Plant cellular and molecular responses to high salinity. Annu.
Rev. Plant Physiol. Plant Mol. Biol., 51, 463–499.
Hay, N. and Sonenberg, N. (2004) Upstream and downstream of
mTOR. Genes Dev., 18, 1926–1945.
Johnson, C.M., Stout, P.R., Broyer, T.C. and Carlton, A.B. (1957)
Comparative chlorine requirements of different plant species.
Plant Soil, 8, 337–353.
Kobayashi, K., Masuda, T., Takamiya, K. and Ohta, H. (2006) Membrane lipid alteration during phosphate starvation is regulated by
phosphate signaling and auxin/cytokinin cross-talk. Plant J., 47,
238–248.
Köck, M., Theierl, K., Stenzel, I. and Glund, K. (1998) Extracellular
administration of phosphate-sequestering metabolites induces
ribonucleases in cultured tomato cells. Planta, 204, 404–407.
Lynch, J. (1995) Root architecture and plant productivity. Plant
Physiol., 109, 7–13.
Ma, Z., Bielenberg, D.G., Brown, K.M. and Lynch, J.P. (2001) Regulation of root hair density by phosphorus availability in Arabidopsis thaliana. Plant Cell Environ., 24, 459–468.
Marschner, H. (1995) Mineral Nutrition of Higher Plants, 2nd edn.
London: Academic Press.
Marschner, H., Kirkby, E. and Cakmak, I. (1996) Effect of mineral
nutritional status on shoot–root partitioning of photoassimilates
and cycling of mineral nutrients. J. Exp. Bot., 47, 1255–1263.
Martin, A.C., del Pozo, J.C., Iglesias, J., Rubio, V., Solano, R., de la
Pena, A., Leyva, A. and Paz-Ares, J. (2000) Influence of cytokinins
on the expression of phosphate starvation responsive genes in
Arabidopsis. Plant J., 24, 559–568.
Menand, B., Desnos, T., Nussaume, L., Berger, F., Bouchez, D.,
Meyer, C. and Robaglia, C. (2002) Expression and disruption of
the Arabidopsis TOR (target of rapamycin) gene. Proc. Natl Acad.
Sci. USA, 99, 6422–6427.
Menand, B., Meyer, C. and Robaglia, C. (2004) Plant growth and the
TOR pathway. Curr. Top. Microbiol. Immunol., 279, 97–113.
Metz, A.M., Timmer, R.T. and Browning, K.S. (1992) Sequences for
two cDNAs encoding Arabidopsis thaliana eukaryotic protein
synthesis initiation factor 4A. Gene, 120, 313–314.
Mimura, T. (1999) Regulation of phosphate transport and homeostasis in plant cells. Int. Rev. Cytol., 149–200.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 545–556
556 Fan Lai et al.
Misson, J., Raghothama, K.G., Jain, A. et al. (2005) A genome-wide
transcriptional analysis using Arabidopsis thaliana Affymetrix
gene chips determined plant responses to phosphate deprivation. Proc. Natl Acad. Sci. USA, 102, 11934–11939.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol.
Plant., 15, 473–497.
Murphy, J. and Riley, J.P. (1962) A modified single solution method
for the determination of phosphate in natural waters. Anal. Chim.
Acta, 27, 31–37.
Nakamura, Y., Awai, K., Masuda, T., Yoshioka, Y., Takamiya, K. and
Ohta, H. (2005) A novel phophatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis. J.
Biol. Chem., 280, 7469–7476.
Narang, R., Bruene, A. and Altmann, T. (2000) Analysis of phosphate
acquisition efficiency in different Arabidopsis accessions. Plant
Physiol., 124, 1786–1799.
Plaxton, W. and Carswell, M. (1999) Metabolic aspects of the
phosphate starvation response in plants. In Plant Responses to
Environmental Stresses: From Phytohormones to Genome
Reorganization (Lerner, H., ed.). New York: Marcel Dekker, pp.
349–372.
van der Ploeg, R., Böhm, W. and Kirkham, M. (1999) On the origin of
the theory of mineral nutrition of plants and the law of the minimum. Soil. Sci. Soc. Am. J., 63, 1055–1062.
Sano, T., Kuraya, Y., Amino, S. and Nagata, T. (1999) Phosphate as a
limiting factor for the cell division of tobacco BY-2 cells. Plant Cell
Physiol., 40, 1–8.
Schmelzle, T. and Hall, M.N. (2000) TOR, a central controller of cell
growth. Cell, 103, 253–262.
Shin, H., Shin, H.-S., Chen, R. and Harrison, M.J. (2006) Loss of
At4 function impacts phosphate distribution between the
roots and the shoots during phosphate starvation. Plant J., 45,
712–726.
Skoog, F. and Miller, C.O. (1957) Chemical regulation of growth and
organ formation in plant tissue cultures in vitro. Symp. Soc. Exp.
Biol., 11, 118–131.
Strawn, D., Doner, H., Zavarin, M. and McHugo, S. (2002) Microscale
investigation into the geochemistry of arsenic, selenium, and iron
in soil developed in pyritic shale materials. Geoderma, 108, 237–
257.
Thornley, J.H. (1972) Balanced quantitative model for root–shoot
ratios in vegetative plants. Ann. Bot., 36, 431–441.
Ticconi, C.A., Delatorre, C.A. and Abel, S. (2001) Attenuation of
phosphate starvation responses by phosphite in Arabidopsis.
Plant Physiol., 127, 963–972.
Ticconi, C.A., Delatorre, C.A., Lahner, B., Salt, D.E. and Abel, S.
(2004) Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint
in root development. Plant J., 37, 801–814.
Tinker, P. and Nye, P. (2000) Solute Movement in the Rhizosphere.
Oxford: Blackwell Scientific.
Wang, H., Qi, Q., Schorr, P., Cutler, A.J., Crosby, W.L. and Fowke,
L.C. (1998) ICK1, a cyclin-dependent protein kinase inhibitor from
Arabidopsis thaliana interacts with both Cdc2a and CYCD3, and
its expression is induced by abscisic acid. Plant J., 15, 501–510.
Werner, T., Motyka, V., Strnad, M. and Schmülling, T. (2001)
Regulation of plant growth by cytokinin. Proc. Natl Acad. Sci.
USA, 98, 10487–10492.
Williamson, L.C., Ribrioux, S.P.C.P., Fitter, A.H. and Leyser, H.M.O.
(2001) Phosphate availability regulates root system architecture
in Arabidopsis. Plant Physiol., 126, 875–882.
Wu, P., Ma, L., Hou, X., Wang, M., Wu, Y., Liu, F. and Deng, X.W.
(2003) Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves. Plant Physiol.,
132, 1260–1271.
Yu, B., Xu, C. and Benning, C. (2002) Arabidopsis disrupted in SQD2
encoding sulfolipid synthase is impaired in phosphate-limited
growth. Proc. Natl Acad. Sci. USA, 99, 5732–5737.
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 545–556