(1 995) 107: 131 3-1 321. Leaf Phosphate Status, Photosynthesis, and. Carbon Partitioning in Sugar Beet. IV. Changes with Time Following lncreased Supply of ...
Plant Physiol. (1995) 107: 1313-1 321
Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet IV. Changes with Time Following lncreased Supply of Phosphate to Low-Phosphate Plants I. Madhusadana Rao' and Norman Terry* Department of Plant Biology, University of California, Berkeley, California 94720 clude (a) increased synthesis of P-free carbon compounds, e.g. starch, SUC,Glc, and cell wall polysaccharides, (b) increased phosphatase activities, and (c) decreased levels of P-containing molecules, e.g. sugar phosphates and adenylates (Rao and Terry, 1989; Rao et al., 1989a, 1990; Terry and Rao, 1991). In vivo experiments using low-P plants have shown that the inhibition of photosynthesis with P deprivation was due to diminished RuBP regeneration rather than to limitations in the supply of ATP and NADPH to the Calvin cycle (Brooks, 1986; Rao et al., 1986, 1990, 1993; Abadia et al., 1987; Brooks et al., 1988; Fredeen et al., 1989, 1990). Unlike short-term P deprivation, which results in the accumulation of starch at the expense of SUC, Suc was accumulated to high levels in P-deficient leaves in addition to starch. We concluded that the changes in carbon partitioning in response to low P were influenced by increases in the relative capacities of the enzymes involved in starch and Suc metabolism (Rao et al., 1990). The objective of the present work was to determine the kinetics of the changes in photosynthesis, carbon partitioning, and growth in response to increasing the Pi supply to low-P-treated plants. In particular, we tested the hypothesis that increased Pi supply to low-P plants will increase leaf RuBP and lead to a rapid recovery of photosynthesis while lowering the pool sizes of the storage carbohydrates, starch, and SUC.The results show that plants subjected to moderate P deficiency and resupplied with phosphate can restore photosynthetic metabolism to control levels within a few hours, that sugar phosphate (especially RuBP) levels increase as starch and SUClevels decrease, and that some effects resulting from P deficiency may persist up to 10 d after P resupply.
Changes in photosynthesis, carbon partitioning, and growth following resupply of orthophosphate (Pi) t o moderately P-deficient plants (low-P) were determined for sugar beets (Beta vulgaris L. cv F58-554H1) cultured hydroponically in growth chambers. One set of plants was supplied with 1.0 mM Pi in half-strength Hoagland solution (control plants), and a second set (low-P plants) was supplied with 0.05 mM Pi. At the end of 2 weeks, the low-P plants were resupplied with 1.O mM Pi. Low-P plants rapidly accumulated large amounts of Pi, and the photosynthesis rate increased t o control values within 4 to 6 h. The rate of photosynthesis appeared to be controlled by ribulose-l,5-bisphosphate (RuBP); low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. Low-P treatment reduced adenylate levels substantially but not nicotinamide nucleotides; adenylate levels recovered t o control values over 3 t o 6 h. With low P, more photosynthate is allocated t o non-P carbon compounds (e.g. starch, sucrose) than to sugar phosphates. When P is resupplied, sugar phosphates increase as starch and sucrose pools decrease; this increase in leaf (chloroplast) sugar phosphates was most likely responsible for the increases in RuBP and photosynthesis and may have increased adenylate levels (through enhanced levels of ribose-5-phosphate).
Phosphorus limitation affects photosynthesis and carbon partitioning differently when applied over long compared to short time periods. Short-term in vitro studies have shown that P deprivation diminishes the export of triose-P from the chloroplast to the cytosol via the Pi translocator; this leads to a buildup of starch and to a decrease in the rate of photosynthesis (see Leegood et al., 1985, for review; Furbank et al., 1987; Giersch and Robinson, 1987). Furthermore, decreases in the export of triose-P from the chloroplast lead to the inhibition of SUCsynthesis in the cytosol (Leegood et al., 1985; Sharkey and Vanderveer, 1989; Stitt, 1989). P deprivation over the long-term initiates adaptive responses that tend to conserve the supply of Pi and thereby maintain high rates of photosynthesis in plants acclimated to P deficiency. These adaptive responses in-
MATERIALS A N D METHODS Plant Culture
Sugar beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers at 25"C, 500 Fmol m-' s-' PFD, and a 16-h photoperiod (Rao and Terry, 1989).
' Present address: Tropical Forages Program, Centro Internacio-
Abbreviations: FBP, fructose-l,6-bisphosphate;F6P, fructosedphosphate; G6P, glucose-6-phosphate; PFD, photon flux density; PG A, 3-phosphoglycerate;RuBP, ribulose-l,5-bisphosphate;Ru5P, ribulose-5-phosphate;triose-P, dihydroxyacetone phosphate plus
nal de Agricultura Tropical, Apartado Aereo 6713, Cali, Colombia, South America. * Corresponding author; e-mail nterryQnature.berke1ey.edu;fax 1-510- 642-4995.
glyceraldehyde-3-phosphate. 1313
1314
Rao and Terry
Low-P and control plants were obtained by growing the plants for 2 weeks at Pi concentrations of 0.05 and 1.0 mM, respectively (Rao and Terry, 1989). P supply to low-P plants was increased by increasing the nutrient solution concentration to 1.0 mM Pi using Ca(H,PO,),. Increased Pi supply to low-P plants was started 2 h into the photoperiod by replacing the nutrient culture medium with freshly prepared nutrient solution of identical nutrient composition for control as well as for low-P plants. The nutrient solutions were changed every week throughout the course of the experiment (1 week of seedling growth with 1mM Pi, 2 weeks of low-P treatment, and 10 d of P resupply treatment). The pH was maintained at about 6.0 by addition of solid CaCO,. Since we used 20-L containers, the composition of nutrient solutions was not altered significantly during the 10-d resupply period. Measurements of gas exchange and of metabolite concentrations of individual leaves were carried out at different intervals after increasing Pi supply to low-P plants. The leaf that had most recently expanded at the time of measurement was chosen for study.
Plant Physiol. Vol. 107, 1995
Assay of Metaholites
Leaf Pi and sugar phosphates (RuBP, PGA, triose-P, F6P, and G6P) were assayed as described before (Rao et al., 1989a).Leaf aclenylates and nicotinamide nucleo tides were assayed according to the methods described by Rao et al. (198913). Starch, SUC,and Glc were estimated as described by Rao et al. (1990). Leaf Area and Dry Weights
Leaf area measurements were made using a Decagon (Decagon Devices, Inc., Pullman, WA) leaf area ineter. The plants were harvested at O, 3, 7, and 10 d after increased Pi supply to low-P plants and were separated into 1.af blades, petioles, storage root, and fibrous roots. The l.issue was dried at 70°C to constant weight. Chemicals
A11 compounds used were purchased from Sigma with the exception of NaHl4CO, (Amersham).
Experimental Design and Statistical Analysis
Plants of the two treatments and 12 sampling times were replicated three times and arranged in a randomized complete block design (using three growth chambers). The data for a11 parameters were analyzed using the analysis of variance procedure of the Statistical Analysis System (SAS/STAT, 1990). Leaf Gas Exchange
The rate of photosynthetic CO, uptake per unit leaf area at 25”C, air levels of CO, (30 Pa) and O, (21 KPa), and growth chamber PFD (500 pmol m-’s-*) was determined using an open-flow gas-exchange system as described previously (Taylor and Terry, 1984). Leaf Sampling and Extraction of Metabolites
RESULTS Leaf Pi Concentration
The leaf blades of plants that had received low P (0.05 mM Pi) for 2 weeks prior to zero time had Pi concentrations that were 88% lower than those of the controls (supplied with 1 mM Pi) (see zero time, Fig. 1).When the P supply to the low-P-treated plants was restored to 1 mM Fi (referred to as P resupply), Pi was translocated to the leaves in massive quantities; blade Pi concentration increased rapidly after resupply began and reached values similar to those of the control leaves within 6 to 8 h (Fig. 1).Seventytwo hours after resupply began, Pi concentration in the leaf blades increased to a maximum of 62 mmol m-” leaf area. Thereafter, Pi concentration decreased progressi vely to 13 mmol m-’ at the end of the growth period (10 d after resupply began). By comparison, the Pi concentration of the control leaf blades decreased from 10 to 5 Inmol m-’
Samples were prepared at O, 3, 6, 24, 30, 48, 54, 75, 101, 144, 192, and 240 h after increased Pi supply to low-P plants. Sampling times of O, 24, 48, 144, 192, and 240 h (O, 1, 2, 6, 8, and 10 d, respectively) after increased Pi supply represent 2 h into the photoperiod. Sampling times of 6,30, and 54 h after increased Pi supply to low-P plants represent 8 h into the photoperiod. Sampling times of 3 and 75 h represent 5 h and 101 h represents 7 h into the photoperiod. Samples were prepared at 25°C and 500 pmol m-’s-* PFD in the growth chamber for the extraction of leaf metabolites in intact leaf tissue. At each time, four leaf discs (3.88 cm2 each) were punched and frozen rapidly in liquid N, (Rao et al., 1989b). Leaf sugar phosphates and Pi were extracted as described by Rao et al. (1989a). Leaf adenylates and nicotinamide nucleotides were extracted as described by Rao et al. (1989b). Leaf starch, SUC,and Glc were extracted as described by Rao et al. (1990). Starch, SUC,and Glc from petioles, storage root, and fibrous roots were extracted from fresh tissue frozen in liquid N,.
I
I
60 -
‘
T
I
I
I
I
1
- 50 40-
E
L- 30 -
Low-P After pi lncrease
LL
10
Control
DAYS AFTER INCREASE IN P; SUPPLY Figure 1. Changes in leaf blade Pi concentration after atldition of Pi to low-P plants (O)and in control plants (O). Values are means -+ SD for three replicaiions.
Effect of lncreased Pi Supply to Low-P Plants
-
^.
I
1315
substantial lowering of the specific leaf weight in the Piincreased plants (Table I). Because of their smaller starting size (and therefore smaller rates of production of photosynthate per plant), the Pi-resupplied plants continued to grow more slowly than the controls (Fig. 2B), and their shoot/root ratio remained low throughout the 10-d growth period (Fig. 2D).
I
Photosynthesis and Leaf Sugar Phosphates
" o
Rates of photosynthesis were measured at irradiances approximating those of the growth chamber in which the plants were grown, i.e. they were measured at 500 pmol m-2 s-l PFD, 30 Pa CO,, and 21 kPa O,. The rate of photosynthetic CO, uptake by leaves of low-P plants (at the zero time) was about 25% lower than the control (Fig. 3; Table 11). This is a typical reduction for low-P sugar beet leaves when photosynthesis is measured under these ambient conditions (Rao and Terry, 1989). Sugar phosphate amounts per area were substantially lower in the low-P leaves at the zero time (Fig. 3; Table 111). RuBP and triose-P in low-P leaves were about 48 and 49% of the control, whereas PGA, F6P, and G6P were 69, 88, and 84% lower, respectively. When additional Pi was supplied to the low-P plants, the rate of photosynthesis recovered to near control values within 4 to 6 h (Fig. 3; Table 11). Three hours after Pi resupply, RuBP and triose-P amounts per area also increased to near or above control values, and PGA, FBP, F6P, and G6P were 50 to 64% of the control (Fig. 3; Table 111). Six hours after resupply, RuBP, triose-P, and FBP were at or above control values, whereas PGA, F6P, and G6P were about 40% below the control (Fig. 3). By 24 h, amounts per area of a11 sugar phosphates were substantially higher than those of the low-P leaves, with triose-P and FBP being much higher than control values (Fig. 3). There were no significant changes with time or treatment in any of these parameters after 24 h.
5 10 5 10 DAYS AFTER INCREASE IN Pi SUPPLY
Figure 2. Changes in total leaf area (A) and dry weight of different plant parts (B-H) after addition of Pi to low-P plants (O) and in controls (O). Values are means ? SD for three replications.
during the 10-d period. Despite the massive amounts of Pi in the leaves of resupplied plants, there were no symptoms of Pi toxicity. Plant Growth
During the period of low-P treatment before P resupply, plants grew more slowly than the controls and had smaller dry weights for a11 plant parts measured (see data at zero time, Fig. 2). Low-P plants had lower shoot/root ratios (Fig. 2d). Leaf area expansion was especially reduced by low P (Fig. 2A) so that specific leaf weights (i.e. weight of blade per leaf area) were considerably higher in low-P leaves than in controls (Table I). When the low-P plants were resupplied with additional Pi, dry weights of plant parts increased progressively with time except for storage root weight, which did not increase until7 d after resupply was initiated (Fig. 2). Leaf area increased more rapidly than leaf blade weight (Fig. 2, A and E), as indicated by the
Leaf Adenylates and Nicotinamide Nucleotides
The amounts per area of adenylates were 60% (or more) lower in leaves of low-P plants compared to the control (zero time, Fig. 4; Table IV). With the exception of NADH, nicotinamide nucleotides were also lower (approximately 20-30% lower, Fig. 4). Three hours after the Pi supply was increased, ATP and ADP increased to 65% of the control, and AMP, NADPH, NADP, and NADH reached control values (Fig. 4). Six hours after resupply, a11 except ADP and NAD had reached control values (Fig. 4; Table IV), and by 24 h, a11 except NADH and AMP had exceeded control
Table 1. fffect of increased Pi supply to low-P plants on specific leaf weights (g m-2) Values are means ? SD for three replications. Days after Pi lncrease
Treatment O
3
7
in
51.4 t- 10.0 45.4 5 11.8
50.5 ? 7.1 44.3 5 10.1
g m-*
Control Low P
+ Pi
43.6 2 7.4 66.6 ? 16.5
55.1 t 10.7 60.8 t 13.6
Rao and Terry
1316
Plant Physiol. Vol. 107, 1995 3 h afier PI addlilon
Figure 3. Changes in photosynthesis (PS), triose-P (TP), and other leaf sugar phosphates in the leaf blade at O, 3 (4 h for PS), 6, and 24 h after Pi addition to low-P plants. Data are expressed as percentages of control at the given time (see tables for absolute values).
150
1111111 Conirol
1 O0
50
O
FS
"PPGA
TF'
BP
F6P
R
06p
I
i00
R W PGA
TF'
BP
F6P
06p
24 h afiar PI addlilon
7
350
I
T
FS
values (Fig. 4). These high levels were maintained for up to 30 h; thereafter, there were no significant changes with time or treatment. Starch, SUC, and Clc
Starch, SUC,and Glc contents in the leaf blades of low-P leaves at the zero time were at least 2-fold more than those of the control (Fig. 5; Table V). Low-P treatment increased starch, SUC,and Glc contents in other parts of the plant as well as leaf blades (Table VI). For example, starch contents were increased 2.8-fold in the leaf petioles, 3-fold in the storage roots, and 5.4-fold in the fibrous roots by low-P treatment (Table VI). SUCand Glc contents were also increased substantially by low-P treatment, but since most of the SUCand Glc was stored in the storage root with much smaller amounts in the petioles and fibrous roots, the most important effect of low P was on storage root SUCand Glc contents (Table VI). Three hours after Pi supply was increased, SUCcontents in leaf blades began to decrease but not starch (Fig. 5; Table V). By 6 h, SUChad decreased to control values and starch Table II. Effect of increased Pi supply to low-P plants on the rate of photosynthesis/area measured at 500 p m o l m-' s-' PFD, 30 Pa CO, and 21 kPa O, Values are means 2 SD for three replications. Rate of Photosynthesis/Area Time after Pi lncrease Control
h O
4 6 24 72-240 (mean)
Low P pmol m-z s-'
14.4 14.2 12.1 13.3 15.6
2 0.2
2 0.2
t 1.7 f 2.8 f 0.9
10.8 13.0 12.0 13.7 14.7
f 1.2 f 1.6 f 1.5 2 0.9 f 0.9
RuBP PGA
TF'
BP
I WP
G6P
had also started to decrease (Fig. 5 ) . By 24 h, starch and Suc reached control values (Fig. 5 ) . High levels of Glc were observed in the P resupply treatment at O, 3, 24, and 48 h after Pi addition (Fig. 5 ) . From 3 to 10 d, there were no significant changes in starch, SUC,or Glc contents in leaf blades of the Pi-increased treatment; however, in the control leaf blades, starch contents increased froni approximately 60 to 135 mmol carbon m-' (data not shown). After 10 d of P resupply, starch, SUC,and Glc contents in other parts of the plant also returned to near control kalues (e.g. in the petiole and storage root, Table VI). In fibi-ous roots, starch and Glc contents decreased to below concrol values during the 10 d of resupply, whereas SUCcclntents increased to 3-fold higher than control values (Table VI). DlSCUSSlON
Pi Transport
The uptake of Pi across the plasma membrane of a plant cell proceeds via 2H+/H,PO,- co-transport driven by an electrochemical proton gradient (Ulrich-Eberius et al., 1984). Under conditions of P deficiency, an eithanced P uptake system may be induced as in spinach (Dietz, 19891, potato (Cogliatti and Clarkson, 1983), barley (Drew et al., 1984), maize, and soybean (Jungk et al., 1990). This enhanced uptake system results in a rapid accumulation of P in leaves once the availability of Pi to roots is improved (Mimura et al., 1990). In the present work, thers was also evidence of a massive uptake of Pi into leaves following P resupply (Fig. 1). The concentration of P in I-esupplied low-P leaves exceeded the control values in about 9 h and reached concentrations 6 times those of the conti-o1in 72 h. The increased activity of the phosphate transport system has been shown to be related to a large increase in V,,, and a significant decrease or no change in the K , of the
Effect of lncreased Pi Supply to Low-P Plants
1317
Table 111. Effect o f increased Pi supply to low-P plants on leaf sugar phosphate amounts per area at O, 3, and 6 h after Pi increase Leaf samples from three replications were pooled for analysis except for RuBP. LSD values are at the P = 0.05 level. NS, Not significant. Leaf Sugar Phosphates
Time after Treatment
Pi lncrease
RuBP
PCA
Triose-P
h
O
Control Low P + Pi
3
Control Low P
6
Control Low P + Pi
66.0 31.7 8.3 77.3 67.7 NS 54.2 65.3 NS
LSD0.05
+ Pi
LSD0.05
LSD0.05
zero tlmt
I
’7
27.3 18.0
18.5 2.3
45.1 7.5
166.1 89.0
13.4 16.8
19.3 9.7
23.1 11.5
47.5 28.5
200.7
13.4 13.4
9.7 14.5
18.5 11.5
45.1 28.5
124.6
ably efficient in transporting Pi to the vacuole (as demonstrated by Mimura et al. [1990] with isolated vacuoles). Photosynthetic Carbon Reduction
Our earlier studies showed that photosynthetic rate was affected relatively little by P deficiency (photosynthesis decreasing by O-33%, depending on light intensity) and that the effect of P deficiency was mediated through a reduction in the rate of RuBP regeneration rather than through an effect on Rubisco activation (Rao and Terry, 1989; Rao et al., 1989a). In the present work, the rate of photosynthesis (measured at 500 pmol m-’s-*) was decreased by 25% and RuBP levels by 60% by low-P treatment. When P was resupplied, photosynthetic rate and RuBP returned to near control values within 3 to 4 h, suggesting that the recovery in photosynthesis was due to a buildup in RuBP levels. There was also a rapid buildup in triose-P (within 3 h), with other leaf sugar phosphates returning to near (or above) control values by the end of 24
I:iuudl 40
ATP
ADP
UIP
wDPn
NADP
6 h
NADH
NAD
ATP
ADP
UIP
NADPH
anel PI addltlon
250
NADP
NADH
NAD
24 h aner PI addltlon
250 T
ATP
C6P
20.6 10.1
Control = 100
I
F6P
124.6 38.0
system (Glass, 1976; Lee, 1982; Lefebvre and Glass, 1982; Cogliatti and Clarkson, 1983; Drew et al., 1984; Jungk et al., 1990). It is generally believed that the activity of ion transport is regulated by some pool of the root or a metabolic product thereof in the cytosol (Glass, 1977; Cram, 1980). When nutrients are abundant, the regulator pool is envisaged as being full and represes the further uptake of the ion in question. When nutrients are in short supply, the size of the regulator pool diminishes and the ion transport system becomes de-repressed increasing ion uptake capacity (such systems are highly ion specific, Lee [1982]).Thus, when Pi was added to low-P plants with a de-repressed ion uptake system, blade Pi concentration increased substantially. After 72 h, blade Pi concentration diminished, suggesting that the enhanced uptake system was becoming repressed; even so, P concentrations in leaves were still substantially higher in resupplied plants after 10 d. It seems likely that much of the increased Pi in P-resupplied plants was located inside the vacuole: the tonoplast has been shown to be remark-
~
FBP
pmol m-’
ADP
UIP
WDPH
NADP
NADH
NAD
ATP
ADP
AMP
wDPn
NADP
NAnn
NAD
nicotinamide nucleotides (NADP = NADP+, NAD = N A D f ) at O, 3, 6, and 24 h after Pi addition to low-P plants. Data are expressed as percentages of control at the given time (see tables for absolute values).
1318
Rao and Terry
Plant Physiol. Vol. 107, 1995
Table IV. Effect of increased Pi supply to low-P plants on leaf adenylate and nicotinamide nucleotide amounts per area at O, 3, and 6 h after Pi increase LSD values are at the P = 0.05 level. NS, Not significant. Time after Pi lncrease
h O
Adenylates Treatment
Control Low P + Pi Control Low P
+ Pi
LSDo.05
6
ADP
Nicotinamide Nucleotides AMP
NADPH
NADP
NADH
NAD
pmol m-'
LSD0.05
3
ATP
Control Low P
+ Pi
LsD0.05
25.7 10.7 0.04 23.3 15.8 1.8 15.7 14.6 0.6
15.8 5.9 7.4 19.8 14.0 NS 18.8 14.0 4.2
h (Fig. 3). Other researchers have found a buildup in sugar phosphates when Pi is resupplied to low-P plants (Brooks, 1986; Dietz and Foyer, 1986; Dietz, 1989). When high levels of Pi (20 mM) were supplied to cut leaves of P-deficient spinach, photosynthesis increased in 5 to 10 min and the level of sugar phosphates (FBP, dihydroxyacetone phosphate, PGA) increased 2-fold (Dietz and Foyer, 1986). Although the resupply of Pi to detached leaves did not increase photosynthetic rates of P-deficient subterranean clover (Bouma, 1975), photosynthetic rates did increase within 24 h of supplying sugar phosphates FBP or G6P. What is the effect of phosphate supply on RuBP formation? The rate of RuBP formation can potentially be affected through ATP supply, through Ru5P kinase activity (Rao and Terry, 1989) or through the supply of Ru5P. In our earlier work we concluded that P deficiency reduced RuBP regeneration primarily through its effect on the pool sizes of the Calvin cycle sugar phosphates, i.e. that sugar phosphate pools were reduced by the action of phosphatases and through increased synthesis of phosphate-free carbon compounds (e.g. starch, SUC)(Rao and Terry, 1989; Rao et al., 1989a, 1990). Under P deficiency, the proportion of fixed carbon going to starch versus RuBP remains the same, with the rates of photosynthesis and starch synthesis both decreasing by about 25% (Rao et al., 1990), but the ratio of sugar phosphates to nonphosphate carbon compounds is substantially reduced. When P is resupplied, starch and SUCcontents decrease as sugar phosphates return to control values. One way this might occur is through the action of adenosine 5-diphosphoglucose pyrophosphorylase. This enzyme is regulated by the ratio of PGA/Pi (Stitt, 1989).When P is resupplied, the PGA/Pi ratio should be very low so that adenosine 5-diphosphoglucose pyrophosphorylase should become less activated and photosynthate (e.g. F6P) going to starch synthesis can be diverted to the buildup of Calvin cycle intermediates and RuBP regeneration. Starch may also contribute to the pool of sugar phosphates through the action of starch phosphorylase. An alternative point of view, which has received support from Jacob and Lawlor (1993), is that variation in P supply controls photosynthesis through the supply of ATP. These researchers presented data showing that ATP content and energy charge limit RuBP production and photosynthesis
10.2 2.2 6.5 5.6 6.9
2.1 1 .8 0.14 1.7 1 .9 NS 2.1 3.3 0.43
NS 7.8 8.8 NS
3.8 2.6 0.1 6 4.1 4.4 NS 4.1 4.4 NS
2.5 2.9 NS 2.1 3.1 NS 2.8 3.5 0.01
10.9 7.5 0.55 13.2 8.9 0.57 13.0 10.0 1.69
in P-deficient sunflower and corn. However, their experimental system differed from ours in that their plants were subjected to more extreme P deficiency: they supplied no Pi in their P-deficient treatment, whereas we induced P deficiency by supplying 0.05 (low-P) compared to 1.0 mM Pi (control). In our research, we concluded that P deficiency
'jkh zo:llllM 3 h afler PI addltlon
zero time
300 T
100
100
Starch
Sucrose
Starch
Glucoae
6 h aner PI addltlon
-
300
200
200
1O 0
O
Ibb Starch
Sucroae
Glucose
30 h aner PI addltlon
I
Starch
Sucrose
G~UCOIM
1 O0
O
Starch
Sucro3e
Glucose
48 h albr PI addltlon
I
I 1
Glucoae
24 h alter PI addltlon
-
300
Sucro3e
7
Sbrch
Sucrose
Glucose
Figure 5. Changes in leaf blade starch, SUC, and Glc ai. O, 3, 6, 24, 30, and 48 h after Pi addition to low-P plants (Clc = 383% of control). Data are expressed as percentages of control at the given time (see tables for absolute values).
Effect of lncreased Pi Supply to Low-P Plants
a11 affect the RuBP regeneration capacity of leaves. At moderate P deficiency conditions, our data indicate that RuBP regeneration in sugar beet leaves may be limited by the supply of Ru5P and/or the initial activity of the Ru5P kinase. The conditions necessary to alter RuBP pool size by this mechanism are not yet clearly understood. Parameters involved in regulation interact and a change in any one of them will result in a change in the activity of the others (Kacser and Burns, 1973; Servaites et al., 1991). When the activity or level of any one of the components is reduced ( e g Ru5P kinase or RuBP), that component temporarily assumes an increased importance until equilibrium is restored. It is the self-regulated lowering of the RuBP pool and not the inability to regenerate it faster that is a major factor in restoring and maintaining metabolic balance (Geiger and Servaites, 1994).
Table V. Effect of increased Pi supply on nonstructural carbohydrate amounts per area of leaf blades at O, 3, 6, and 24 h after Pi increase LSD values
are at the P = 0.05 level. NS, Not significant. Nonstructural Carbohydrates
Time after
Pi lncrease
Treatment
Starch
h
Control Low P
3
Control Low P
19.3 52.7 16.7 26.8 71.5 8.2 71.1 102.8 24.8 43.5 50.6 4.9
+ Pi
LSD0.05
+ Pi
LSD0.05
24
Clc
mmol C m-z
O
6
suc
Control Low P + Pi LsD0.05 Control Low P Pi
+
LSDn nc
1.5 3.7 1.8 1.8 3.3 0.6 3.6 3.6 NS 2.6 3.3 NS
1319
0.2 0.6 0.2 0.3 1 .o 0.3 0.6 0.8 NS 0.4 1.1 0.3
Carbon Partitioning and Allocation
Plants respond to P stress by altering the partitioning of photosynthate among plant parts and by altering the allocation of carbon to starch, SUC,and other nonstructural carbohydrates, and to structural carbohydrates such as cellulose and hemicelluloses (Rao et al., 1990). Low-P treatment restricts the growth of the shoot, especially the area expansion of leaves, but has only minor effects on photosynthate production per area. This results in an increased availability of carbohydrates for root growth, which may b e enhanced relative to shoot growth (Ulrich and Berry, 1961; Christie and Moorby, 1975; Clarkson and Scattergood, 1982; Cogliatti and Clarkson, 1983; Foyer and Spencer, 1986; Sicher and Kremer, 1988; Fredeen et al., 1989; Rao and Terry, 1989; Adu-Gyamfi et al., 1990; Khamis et al., 1990; Rao et al., 1990; Jacob and Lawlor, 1991; Usuda and Shimogawara, 1991; McArthur and Knowles, 1993). With restoration of the P supply, less photosynthate was partitioned to storage roots, which virtually ceased growth for 7 d after P resupply (Fig. 2G). Furthermore, leaves expanded more rapidly than they accumulated dry weight (specific leaf weight decreased), but despite the relative
did not affect RuBP regeneration through effects on the photosynthetic electron system and/or ATP supply. This view is supported by the following: (a) our earlier work showed that low P had no major effect on the structure and function of the photosynthetic electron transport system or on photosynthetic quantum yield (Abadia et al., 1987) and (b) ATP supply did not appear to be limiting RuBP regeneration because low-P treatment increased the ratios of triose-P/PGA, ATP/ADP, and NADPH/NADP+ (Rao et al., 1989a). The latter finding was repeated in the present work. We believe that on P resupply the primary event is a buildup in sugar phosphates, which leads to increased RuBP and photosynthesis, as well as to increased ribosedphosphate, which is needed for the synthesis of the adenylate precursor IMP (Rao et al., 1989a). Thus, the synthesis of ATP and other adenylates may be secondary to the buildup in sugar phosphates (ATP takes 6 h to reach control values compared to 3 h for RuBP and triose?, see Fig. 4). The availability of fixed carbon, the initial activity of the Calvin cycle enzymes, and the supply of ATP and NADPH
Table VI. Effect of increased Pi supply on nonstructural carbohydrates of petioles, storage root, and fibrous roots LSD values are at the P = 0.05 level. NS, Not significant. Days after Pi lncrease
Plant Part
Treatment
10
O
Starch
suc
Clc
Starch
SUC
Clc
pmol Cg-' fresh wt
Petiole
Control Low P
Storage root
Control Low P
+ Pi
LSD0.05
+ Pi
LSD0.05
Fibrous roots
Control Low P
+ Pi
LSD0.05
14.7 41.8 16.4 25.3 75.7 16.9 4.6 24.5 16.4
15.5 27.3 NS 974 1814 652 86.9 34.2
NS
0.4 10.8
NS 10.4 82.8 16.3 1.3 10.5 NS
60.2 56.8
NS 33.6 57.0 NS 26.7 9.0 1.7
46.2 41.5 NS 21 37 1622 196 49.9 156 26
11.9 6.5 NS 24.1 24.9 NS 1.2 0.5 NS
1320
Rao and Terry
increase in leaf expansion, resupplied .plants continued to partition less photosynthate to shoots than roots (compared to control plants) throughout the 10 d of resupply (Fig. 2D). In leaf blades of plants subjected to P deficiency, there was a substantially increased allocation of photosynthate to the nonstructural carbohydrates, starch, SUC,and Glc (and probably also to structural carbohydrates such as cellulose and hemicellulose, Rao et al. [19901), whereas sugar phosphates in the leaf blade substantially decreased. Similar large increases in starch, SUC,and Glc occurred in other parts of P-deficient plants, i.e. petioles, storage roots, and fibrous roots. On P resupply, the allocation of carbohydrates in leaf blades changed rapidly, and by 24 h starch and Suc contents had decreased and sugar phosphates increased to near control values. After 10 d, starch, SUC,and Glc contents in petioles and storage roots were at control values, but in fibrous roots the allocation of carbohydrates shifted from starch and Glc to SUC;Suc contents in the fibrous roots were lower in low-P plants than in the control but, after 10 d of resupply, increased to values 3-fold higher than the control (Table VI).
CONCLUSIONS Probably the most striking event to occur when Pi is resupplied to low-P plants is the rapid and massive uptake of Pi into plant tissues: in leaf blades, Pi concentration reached values 6 times those of the control leaves in 72 h. The photosynthesis rate of low-P leaves recovered to control values 4 to 6 h after P resupply. The rate of photosynthesis appeared to be controlled by RuBP levels in relation to variation in P supply; low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. The relation between phosphorylated photosynthate (sugar phosphates) and non-P carbon compounds seems to be pivotal with respect to P supply: under low P, more photosynthate is allocated to non-P carbon compounds (starch, SUC,cell wall polysaccharides, etc.) than to sugar phosphates. This adaptive feature of P-deficient plants is instrumental in "freeing up" Pi to maintain rapid rates of photosynthesis; despite an 88% reduction in leaf blade P concentration, low-P leaves were able to sustain rates of photosynthesis, which were 75% of the control. On P resupply, sugar phosphates increase as starch and Suc pools decrease. This increase in leaf (chloroplast) sugar phosphates may have been responsible for the buildup in the levels of ribose-5-phosphate and ATP (and other adenylates), as well as RuBP and photosynthesis, on P resupply. Although many effects of low-P treatment were reversed within 24 h of P resupply, some effects of low-P treatment may persist u p to 10 d; for example, in P-resupplied plants, storage roots ceased growth for 7 d, shoot/root ratios remained low throughout the experimental period, but fibrous root Suc concentrations increased 3-fold during the 10-d period.
Plant Physiol. Vol. 107, 1995 ACKNOWLEDGMENTS
We thank C. Carlson for his help with culturing sugar beet plants ancl carrying out gas-exchange analysis and DI,.A.R. Arulanantham for help with the metabolite assays.
Received July 26,1994; accepted December 6,1994. Copyright Clearance Center: 0032-0889 / 95 / 107/ 1313,'09.
LITERATURE ClTED
Abadia J, Rao IM, Terry N (1987) Changes in leaf phosphate status have only small effects on the photochemical appara tus of sugar beet leaves. Plant Sci 50: 49-55 Adu-Gyamfi JJ, Fujita K, Ogata S (1990) Phosphorus fractions in relation to growth in pigeon pea (Cujunus cujan (L) Millsp.) at various levels of P supply. Soil Sci Plant Nutr 36: E31-543 Bouma D (1975) Effects of metabolic phosphorus cornpounds on rates of photosynthesis of detached phosphorus-deficient subterranean clover leaves. J Exp Bot 26: 52-59 Brooks A (1986) Effects of phosphorus nutrition on ribulose-1,5bisphosphate carboxylase activation, photosyntheiic quantum yield and amounts of some Calvin-cycle metabolites in spinach leaves. Aust J Plant Physiol 13: 221-237 Brooks A, Woo KC, Wong SC (1988) Effects of phosphorus nutrition on the response of photosynthesis to CO, and O,, activation of ribulose bisphosphate carboxylase and amounts of ribulose bisphosphate and 3-phosphoglycerate in spinach leaves. Photosynth Res 15: 133-141 Christie EK, Moorby J (1975) Physiological responses of semi-arid grasses. 1. The influence of phosphorus supply on growth and phosphorus absorption. Aust J Agric Res 26: 423-436 Clarkson DT, Scattergood CB (1982) Growth ancl phosphate transport in barley and tomato plants during the development of, and recovery from, phosphate-stress.J Exp Bot 33: 865-875 Cogliatti DH, Clarkson DT (1983) Physiological changes in and phosphate uptake by potato plants during development of and recovery from phosphate deficiency. Physiol Plant 58: 287-294 Cram WJ (1980) A common feature of the uptake of solutesby root parenchyma cells. Aust J Plant Physiol 7: 4 1 4 9 Dietz K-J (1989) Recovery of spinach leaves from sulfate and phosphate deficiency. J Plant Physiol 134: 551-557 Dietz K-J, Foyer C (1986) The relationship between phosphate status and photosynthesis in leaves; reversibility of the effects of phosphate deficiency on photosynthesis.Planta 16'7: 376-381 Drew MC, Saker LR, Barber SA, Jenkins W (1984) Changes in the kinetics of phosphate and potassium absorption in nutrientdeficient barley roots measured by a solution-de]~letiontechnique. Planta 160: 490-499 Foyer C, Spencer C (1986) The relationship between phosphate status and photosynthesis in leaves. Effects on intiacellular orthophosphate distribution, photosynthesis and assimilate partitioning. Planta 167 369-375 Fredeen AL, Raab TK, Rao IM, Terry N (1990) Effwts of phosphorus nutrition on photosynthesis in Glycine mrix (L) Merr. Planta 181: 399-405 Fredeen AL, Rao IM, Terry N (1989) Influence of phosphorus nutrition on growth and carbon partitioning in Glycine max. Plant Physiol 8 9 225-230 Furbank ET, Foyer CH, Walker DA (1987) Regulatim of photosynthesis in isolated spinach chloroplasts during, orthophosphate limitation. Biochim Biophys Acta 87: 552-56 1 Geiger DR, Servaites JC (1994) Diurna1 regulation of photosynthetic carbon metabolism in C, plants. Annu Rev I'lant Physiol Plant Mo1 Biol45: 235-256 Giersch C, Robinson SP (1987) Regulation of photosynthetic carbon metabolism during phosphate limitation of ph otosynthesis in isolated spinach chloroplasts. Photosynth Res 111:211-227 Glass ADM (1976) Regulation of potassium absorptlon in barley roots. An allosteric model. Plant Physiol 5 8 33-37
Effect of lncreased Pi Supply to Low-P Plants
Glass ADM (1977) Regulation of Kf influx in barley roots: evidente for direct control by nutrient KC. Aust J Plant Physiol 4 313-318 Jacob J, Lawlor DW (1991) Stomatal and mesophyll limitations of photosynthesis in phosphate deficient sunflower, maize and wheat plants. J Exp Bot 42: 1003-1011 Jacob J, Lawlor DW (1993) In vivo photosynthetic electron transport does not limit photosynthetic capacity in phosphate deficient sunflower and maize leaves. Plant Cell Environ 16: 785-795 Jungk A, Asher CJ, Edwards DG, Meyer D (1990) Influence of phosphate status on phosphate uptake kinetics of maize (Zea mays) and soybean (Glycine ma).Plant Soil 124: 175-182 Kacser H, Burns JA (1973) The control of flux. Symp SOCExp Biol 27: 65-104 Khamis S, Chaillou S, Lamaze T (1990) CO, assimilation and partitioning of carbon in maize plants deprived of orthophosphate. J Exp Bot 41: 1619-1625 Lee RB (1982) Selectivity and kinetics of ion uptake of barley plants following nutrient deficiency. Ann Bot 5 0 429-449 Leegood RC, Walker DA, Foyer CH (1985) Regulation of the Benson-Calvin cycle. In J Barber, NR Baker, eds, Photosynthetic Mechanisms and the Environment. Elsevier, Amsterdam, The Netherlands, pp 189-258 Lefebvre DD, Glass ADM (1982) Regulation of phosphate influx in barley roots: effects of phosphate deprivation and reduction of influx with provision of orthophosphate. Physiol Plant 54: 199-206 McArthur DJ, Knowles NR (1993) Influence of vesicular-arbuscular mycorrhizal fungi on the response of potato to phosphorus deficiency. Plant Physiol 101: 147-160 Mimura T, Dietz K-J, Kaiser W, Schramm MJ, Kaiser G, Heber H (1990) Phosphate transport across biomembranes and cytosolic phosphate homeostasis in barley leaves. Planta 180: 139-146 Rao IM, Abadia J, Terry N (1986) Leaf phosphate status and photosynthesis in vivo: changes in light scattering and chlorophyll fluorescence during photosynthetic induction in sugar beet leaves. Plant Sci 44: 133-138 Rao IM, Arulanantham AR, Terry N (1989a) Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet. 11. Diurnal changes in sugar phosphates, adenylates, and nicotinamide nucleotides. Plant Physiol 9 0 820-826
1321
Rao IM, Arulanantham AR, Terry N (1989b) Diurnal changes in adenylates and nicotinamide nucleotides in sugar beet leaves. Photosynth Res 23: 205-212 Rao IM, Fredeen AL, Terry N (1990) Leaf phosphate status, photosynthesis and carbon partitioning in sugar beet. 111. Diurnal changes in carbon partitioning and carbon export. Plant Physiol 9 2 29-36 Rao IM, Fredeen AL, Terry N (1993) Influence of phosphorus limitation on photosynthesis and carbon allocation and partitioning in sugar beet and soybean grown with a short photoperiod. Plant Physiol Biochem 31: 223-231 Rao IM, Terry N (1989) Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet. I. Changes in growth, gas exchange, and Calvin cycle enzymes. Plant Physiol 9 0 814-819 SASlSTAT (1990) SAS/STAT User’s Cuide, version 6. SAS Institute Inc., Cary, NC Servaites, JC, Shieh W-J, Geiger DR (1991) Regulation of photosynthetic carbon reduction cycle by ribulose bisphosphate and phosphoglyceric acid. Plant Physiol 97: 1115-1121 Sharkey TD, Vandeweer PJ (1989) Stromal phosphate concentration is low during feedback limited photosynthesis. Plant Physiol 91: 679-684 Sicher RC, Kremer DF (1988) Effects of phosphate deficiency on assimilate partitioning in barley seedlings. Plant Sci 57: 9-17 Stitt M (1989) Photosynthetic carbon partitioning: its regulation and possibilities for manipulation. Physiol Plant 77: 633-641 Taylor SE, Terry N (1984) Limiting factors in photosynthesis. V. Photochemical energy supply colimits photosynthesis at low values of intercellular CO, concentration. Plant Physiol75 82-86 Terry N, Rao IM (1991) Nutrients and photosynthesis: iron and phosphorus as case studies. In JR Porter, DW Lawlor, eds, Plant Growth: Interactions with Nutrition and Environment. Cambridge University Press, Cambridge, UK, pp 55-79 Ulrich A, Berry WL (1961)Critical phosphorus leve1 for lima bean growth. Plant Physiol 3 6 626-633 Ulrich-Eberius CI, Novacky A, van Bel AJE (1984) Phosphate uptake in Lemna gibba G1: energetics and kinetics. Planta 161: 46-52 Usuda H, Shimogawara K (1991)Phosphate deficiency in maize. I. Leaf phosphate status, growth, photosynthesis and carbon partitioning. Plant Cell Physiol 3 2 497-504
