Journal of Experimental Botany, Vol. 47, No. 297, pp. 497-505, April 1996
Journal of
Experimental
Botany
Phosphorus responses of C3 and C4 species
Marilou Halsted and Jonathan Lynch1
Department of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA
Received 14 November 1994; Accepted 7 December 1995
Abstract
An hypothesis was formulated that phosphorus (P) partitioning in tissues of C4 leaves would permit C4 plants
to resist P deficiency better than C3 plants. To test
this hypothesis, 12 C3, C4, and C3-C4 intermediate
species were grown at adequate, deficient, and
severely deficient P supply in a solid-phase-buffered
sand culture system to characterize photosynthetic
and growth responses. Species differed considerably
in response to P stress. The growth of C3 species was
more sensitive to P supply than C4 species, but C3 and
C4 species had similar photosynthetic P use efficiency,
and C4 species did not have low leaf P content, contrary to our hypothesis. In fact, leaf photosynthetic
rates were not correlated with growth responses.
Moncots had lower leaf P content and better maintenance of leaf production under P stress than dicots,
because of greater inhibition of branching (dicots) than
of tillering (monocots). The most P efficient species in
this survey was Brachiaria, a C4 monocot that
increased root biomass allocation under stress while
maintaining P allocation to the shoot. It is concluded
that C4 species are not inherently more P efficient
than C3 species, but that monocots are more P efficient
than dicots, because of contrasting P and biomass
allocation under stress.
Key words: Phosphorus deficiency, C3 plants, C4 plants,
growth response.
Introduction
It has been proposed that C 4 plants originated in the
tropics and subtropics where high light, low water availability and high temperatures favoured the selection of
plants better adapted to these conditions (Bjorkman,
1971; Black, 1973). Distribution patterns of North
American Graminae support this idea (Terri and Stowe,
1
To whom correspondence should be addressed. Fax: +1 814 863 6139.
Oxford University Press 1996
1976), but question the significance of aridity as a selective
pressure. Many tropical soils are highly weathered and
have low P availability (Sanchez, 1976), so it is possible
that nutrient stress also acted as a selective pressure for
the adaptation of C 4 plants.
There is precedence for investigating the differences in
nutrient use efficiencies of C 3 and C 4 species. Brown
(1978) speculated that C 3 and C 4 species differ in their
nitrogen ( N ) use efficiencies. Sage and Pearcy (1987a, b)
investigated this idea and found that photosynthetic parameters and growth must be considered together since,
while a C 4 species seemed to be more efficient at carbon
fixation during N stress, its growth patterns were less
efficient than those of a C 3 analogue.
There is indirect evidence to suggest that C 3 and C4
plants differ in both photosynthetic and growth responses
to low P environments. A persistent problem of legumebased tropical pasture systems, where low P soils predominate, is the aggressiveness of C 4 grasses, which outcompete C 3 leguminous species (Sanchez, 1976). In the
temperate zone, Morris et al. (1982) observed that warm
season grasses (C 4 ) had higher yield with a lower tissue
P concentration than cool season grasses (C 3 ) on a low
P soil.
Evidence for differing C 3 and C 4 cellular responses to
P stress is indicated by an increase in leaf starch content
of C 3 plants during P stress (Portis, 1982; Dietz and
Heilos, 1990; Fredeen et al., 1989) that can be correlated
to changes in enzymes of the starch biosynthetic pathway
(Fredeen et al., 1989). Earlier work showed a decrease in
starch synthesis in C 4 leaf discs compared to an increase
in starch analysis in C 3 leaf discs (HeroId et al., 1976)
during P stress. More recently, Usuda and Shimogawara
(1991) using maize, a C 4 species, measured a decrease in
partitioning to starch in a long-term study under P stress.
The effect of P deficiency on the specific activity of
Rubisco, the ratio of 3-phosphoglycerate to RuBP, and
the ratio of the stromal concentration of RuBP to that
of RuBP-binding sites differed between sunflower (C3)
498 Halsted and Lynch
and maize (C4) (Jacob and Lawlor, 1992). The recent
observation that P deficiency decreases the in vivo CO2/O2
specificity factor of Rubisco in intact leaves of sunflower
(Jacob and Lawlor, 1993), resulting in relatively more
photorespiration, also suggests that the photosynthetic
responses of C3 and C4 plants may differ.
The Kranz anatomy of C4 plants consists of welldeveloped bundle sheath cells that surround the vascular
bundle. One characteristic of these cells is that they have
a higher concentration of organelles than the mesophyll
cells. The phosphate/triose-phosphate translocator located on the chloroplastic inner envelope membrane
depends on cytoplasmic inorganic phosphate (P;) concentration to keep the flow of exported assimilates and
imported P, in balance (Herold and Walker, 1979). When
cytoplasmic P, is deficient there is a reduction in triosephosphate transport out of the chloroplast (Herold and
Walker, 1979), increased levels of triose-phosphates
within the chloroplast that can result in starch formation
(Heldt et al., 1977; Herold et al., 1976) and, eventually,
feedback inhibition of carbon assimilation (Heldt et al.,
1977; Herold and Walker, 1979), perhaps as a result of
decreased ribulose-bisphosphate regeneration (Jacob and
Lawlor, 1992). Inorganic P is also required for PEP
export in mesophyll chloroplasts of C4 plants (Huber and
Edwards, 1977).
It was hypothesized that C4 plants may be able to
maintain photosynthesis at bulk leaf P levels inadequate
for C3 plants, by preferential allocation of leaf P to the
bundle sheaths. Maintenance of cytoplasmic P; concentration in the cytoplasm of bundle sheath cells would permit
continued C fixation and export by the majority of leaf
chloroplasts, despite reductions in cellular P concentration
in surrounding mesophyll cells.
As a first step towards the evaluation of this hypothesis,
and to confirm indirect evidence of differences in the P
efficiency of C3 and C4 plants, the objective of this study
was to determine differences in photosynthetic phosphorus use efficiency among C3, C4 and C 3 -C 4 intermediates under realistic P regimes.
heated and cooled as needed to maintain reasonable growth
conditions, the three experiments varied in light intensity,
photoperiod, and average temperature. The results presented
here are primarily from the second experiment since this
experiment surveyed the largest number of species in the
P-buffered, sand/alumina system; more detailed accounts of
experiments 1 and 3, which corroborate the results presented
here, are described by Halsted (1994).
Plant materials and culture
Stock seed and/or cuttings of different species were obtained
from a variety of sources and propagated to generate sufficient
experimental material (Table 1). Seeds of Phaseolus and maize
were surface-sterilized then germinated on germination paper
soaked in 0.5 mM CaSO 4 . Phaseolus seed was scarified prior to
germination. Amaranthus tricolor was sown in river sand, while
other seeded species were sown in soilless media. Cuttings for
all experiments had been taken from vegetative stems 3-4 weeks
prior to transplant and rooted without hormone in soilless
media. All roots were rinsed with distilled water to remove
media prior to transplant. Plant material was grown with
adequate nutrition prior to transplant. Experiments were
conducted in a greenhouse at Penn State University (40° 85' N,
77°83'W).
Seedlings or rooted cuttings were transplanted into 20 1 pots
in a solid-phase-buffered sand culture system (Lynch et al.,
1990) between 10 May and 18 May 1993. There was one plant
per pot except for Alloteropsis species, which were so small that
three plants per pot were planted and then thinned. Plants were
grown with a maximum daytime temperature of 29 °C and a
minimum night-time temperature of 18°C. Natural photosynthetically active radiation {PAR) varied throughout the day
with a maximum of 1800 fimol m " 2 s~'. Media temperature
during the day at 13 cm below the surface was 20 °C. The solidphase system provided three buffered P treatments: 1 fiM
(Low); 3-5/xM (Medium); and 15/*M (High). All other
nutrients were in adequate supply and present in the following
concentrations (inmmolm" 3 ): 1500 KNO 3 , 1200 Ca(NO 3 ) 2 ,
400NH 4 NO 3 , 800MgSO 4 , 300 K 2 SO 4 , 300 (NH 4 ) 2 SO 4 , 1.5
MnSO 4 , 1.5ZnSO4, 0.5 CuSO 4 , 5.0 Fe-NaEDTA, 0.143
(NH 4 ) 6 Mo 7 O 24 , 0.5 Na 2 B 4 O 7 . Panicum species displayed iron
deficiency symptoms, which were alleviated with the application
of foliar chelated iron. Pots were irrigated with nutrient solution
as needed, approximately twice daily. Neither Rhizobium nor
mycorrhizas were employed, in order to avoid the additional
complexity of genetic differences in symbiotic efficiency. In an
earlier experiment with Phaseolus in this type of system, the
effects of VAM symbiosis and additional P on plant growth
and photosynthesis were similar (Lynch et al., 1991).
Materials and methods
Experimental design
A variety of C 3 and C 4 species was surveyed to minimize the
possibility that the results would be biased by the adaptation
of any particular C 3 or C 4 species to the growth conditions.
Comparing one or two species was a less desirable option
because of the chance of environmental conditions favouring
one species over another. Three experiments were performed in
a greenhouse under different environmental conditions. The
first experiment used a soilless medium which did not buffer P
and was conducted from July to October, 1992. The second
experiment, which is the focus of this paper, utilized a
sand/alumina medium which provided solid-phase P buffering
and was conducted from May to July, 1993. The third
experiment was also conducted in the P-buffered system from
October and November, 1993. Although the greenhouse was
Each species was grown at each P treatment level. Replicates
(w = 3-4) were planted and measured several days apart to
minimize the effect of daily differences in environmental
conditions on gas exchange parameters. Plant/treatment positions were arranged in a randomized complete block design,
with time of planting as the block.
Leaf gas exchange
Carbon dioxide exchange rate (CER) was measured with a
LI-COR 6200 Portable Photosynthesis System (Lincoln, NE).
The youngest fully expanded leaf of an individual plant was
enclosed in the LI-COR leaf chamber and leaf CER was
monitored for 45 s at saturating light. A fan positioned several
feet from the chamber helped to maintain chamber and leaf
C3/C4 phosphorus responses
499
Table 1. Information about species used in the experiment
Plants were grown for 7.5 weeks in stressful (Low P) or control (High P) conditions.
Species
Photosynthetic Anatomical
type
class
Synonum
used in
figures
Growth
Supplier
Alloteropsis semialata
eikloniana (R.Br.)
Hitchcock
Alloteropsis semialata
semialata (R.Br.)
Hitchcock
Amaranthus tricolor L.
Brachiaria dictyoneura
(Fig. and De Not.)
Stapf.
Flaveria floridana J. R.
Johnston
Flaveria pringlei Gandoger
C3
Monocot
ASE
Perennial
Grassland Research Centre Seed
Genebank, South Africa
C4
Monocot
ASS
Perennial
Grassland Research Centre Seed
Genebank, South Africa
3
C4
C4
Dicot
Monocot
AT
BD
Annual
Perennial
Johnny's Seed
CIAT/6133
Seed
Crown
separation
2
1
C3-C4
Dicot
FF
Perennial
Cutting
4-8
C3
Dicot
FP
Perennial
Cutting
4-8
Flaveria trinervia Mohr
C4
Dicot
FT
Perennial
Panicum laxum Mez.
Panicum miliaceum L.
Panicum mihoides Neese ex
Trin
Phaseolus vulgaris L.
Zea mays L.
C3
C4
C 3 -C 4
Monocot
Monocot
Monocot
PL
PMIA
PMIO
Annual
Annual
Annual
R. H. Brown, Univ.
Georgia
R. H. Brown, Univ.
Georgia
R. H. Brown. Univ.
Georgia
USDA/496383
Du Pont
USDA/285219
Seed
Seed
Seed
3
1
3-9
C3
C4
Dicot
Monocot
PV
ZM
Annual
Annual
CIAT/G-4000
Pioneer/Hybrid 3394
Seed
Seed
Primary leaves
Primary leaf
temperatures without mechanically stressing the plant. Relative
humidity (RH) within the chamber varied less than 2% during
measurement. On cloudy days, leaves were acclimated for
2-4 min to saturating light (1300-1800 ^mol m" 2 s~' provided
by a metal halide spotlight) prior to measurement. Light curves
had been constructed to determine saturating light levels for
each species illuminated in this way. One measurement was
taken from each plant. All measurements were made between
09.30 h and 11.45 h and between 13.00 h and 15.30 h on a given
data collection day and plants were selected randomly for
measurement so that no species or treatment were biased by
time of day. Data are presented from both sampling periods,
which were not significantly different.
Following the CER measurements the assay leaf was marked
and the leaf area inside the chamber was traced on paper, cut
out and the area measured on a LI-COR Model 3100 leaf area
meter. Assayed leaf area was then excised, weighed and dried
at 60 °C for at least 48 h in a forced air drier and ashed for 6 h
at 475 °C. Ash was then dissolved in 0.1 N HC1 and leaf total
P content was measured using a colorimetric assay (Murphy
and Riley, 1962).
Growth measurements
At 52 d after transplanting, plant height, stem width and
number of branches were measured for the dicots while height
of the tallest tiller (measured from base to uppermost leaf
ligule), and tiller number were measured for the monocots.
Shoots were harvested and divided into leaves, stems, and
flowers (where appropriate) for biomass determination. Roots
were extracted from the buckets and rinsed in distilled water to
remove media, blotted dry and weighed. Total leaf area was
measured directly or estimated with subsamples. Tissues were
dried for at least 48 h at 60 °C in a forced air oven. Plant parts
were ground in a Wiley Mill through a 40 mesh screen. Dried,
ground tissue was then ashed at 475 °C for 6 h. dissolved in
Propagation
Initial number
of leaves or
tillers
Seed, cutting 4-12
0.1 N HC1 and assayed for total P with the colorimetric assay
as above.
In a later experiment plant roots were extracted from the
medium and a subsample was stored in 10% isopropanol. Root
length was measured with Tennant's line-intersect method
(Tennant, 1975); dry weight was determined by drying the
subsamples in a forced air oven at 60 °C for at least 24 h. The
width of the main roots was measured with calipers.
Plants grown with the high P treatment were considered
control plants. Data from plants grown with the low and
medium P treatments were compared with average control plant
values for a given growth parameter to generate the percentage
of control values shown in Fig. 3.
Statistical analysis
Three photosynthetic types and two effective P levels, high and
low (which included both the medium and low P treatments,
see 'Results' section), and the interaction between photosynthetic type and P level were included as independent variables
in a 3 x 2 factorial model. The a priori comparisons were tested
by means of orthogonal contrasts. All statistical analyses were
performed using SYSTAT 5.2.1 (Wilkinson, 1989). Residual
diagnostics suggested that data were non-normal so all data
were In transformed, unless otherwise noted, prior to ANOVA.
Mean values presented are least-squared estimates of true means
from the ANOVA model, and so may not always appear to be
arithmetically orthogonal in the tables.
Results
Diagnosis and visual characteristics of P stress
The solid-phase, phosphorus-buffered, sand culture
system was effective at producing stable control and P
stress conditions; the P stress treatment level was low
500
Halsted and Lynch
enough that all species experienced P stress. High tissue
P concentration (Halsted, 1994), as well as visual observation, confirmed that the control plants were not P-stressed.
Panicum miliaceum was not included in the analysis
because it flowered and senesced within days after transplanting; Zea mays was not included because an interveinal chlorosis was evident in all plants. This species also
grew much more quickly than the others, became potbound and was harvested before the end of the experiment. The low and medium P treatment data were not
significantly different from one another in any parameter
so the data has been pooled and presented as one 'Low
P' treatment.
Phosphorus-stressed plants had less biomass, fewer
branches or tillers, fewer leaves, reduced leaf size, and
fewer, if any, inflorescences, compared with control
plants. Growth responses to P stress varied greatly among
species. Dry weights of plants parts are shown in Table 2;
other growth analyses can be found in Halsted (1994).
Pigment changes were also noted in the low P plants of
some species. All Flaveria species showed anthocyanin
accumulation in stems and older leaves during P stress.
Unstressed Amaranthus tricolor was characterized by
leaves that were purple in the centre and green on the
leaf margin. The ratio of purple to green areas was larger
in P-stressed A. tricolor. Phaseolus developed darker green
leaves when P stressed. The monocots, except for maize,
did not show any anthocyanin accumulation except at
leaf tips just prior to leaf senescence. AHoteropsis semialata
eikloniana (C3) leaves were darker green at low P, but
the leaves of the C4 ecotype, A. semialata semialata, were
not. Stressed Brachiaria dictyoneura leaves showed neither
anthocyanin accumulation nor greening. Leaf senescence
was accelerated in P-stressed plants.
Figure 1 compares the growth of all species under both
stressful and control P conditions. Species that are sensitive to P stress relative to other species will lie in the lower
right corner of the figure indicating that they perform
well with enough P but poorly with limited P. These
species can also be considered responsive since they
respond to changes in P nutrition. Species with lower
sensitivities will lie in the upper left corner indicating that
with adequate P they will not perform as well as responsive species, but during P stress they will perform better
than the responsive species. It is interesting to note that
while there is substantial overlap in the two groupings of
C3 and C4, the most responsive/sensitive species are C3
while the least responsive/sensitive are C4. Intermediate
species group together in the overlap and closely related
species have similar sensitivities. Similar results were
obtained in the preliminary study that had been conducted
at a different time of year with a non-buffered P supply.
C3 plants were larger at high P than the C4 plants
(Table 3) due both to greater root and greater shoot
mass, resulting in similar root-to-shoot ratios. The C3
and C4 plants had similar average biomass production
under P stress but the root-to-shoot ratio was higher in
C3 plants. In addition, the increase in root-to-shoot ratio
caused by P stress was greater in C3 plants.
Photosynthetic phosphorus use efficiency
Photosynthetic phosphorus use efficiency (PPUE) ranged
from 54 to 374jnmol C O j S ^ g " 1 P among species
(Table 4). C3 and C4 plants had similar mean PPUEs
(Fig. 2), but C3 PPUE was significantly higher than
C 3 -C 4 PPUE. Slight differences within photosynthetic
types and classes resulted from variability in leaf P content
(Fig. 2) rather than CER (Fig. 2). For example, the
greater PPUE of C3 species compared to C 3 -C 4 is due to
a 2-fold lower leaf P content.
Table 2. Growth parameters of species grown under P deficiency (Low P) or adequate P (High P) in a P-buffered medium
High P values are means of 2-4 replicates; low P values are means of 6-8 replicates."
Dry weight per plant (g)
Total
High P
AHoteropsis semialata
eikloniana
A lloteropsis semialata
semialata
Amaranthus tricolor
Brachiaria dictvoneura
Flaveria floridana
Flaveria pringlei
Flaveria trinervia
Panicum laxum
Panicum milioides
Phaseolus vulgaris
Root
Shoot
Low P
High P
Root shoot
i
Low P
High P
Low P
High P
Low P
1.9(0.1)
0.59(0.14)
1.4(0.2)
0.29(0.05)
0.46 (0.03)
0.30(0.11)
0.33(0.03)
0.95 (0.27)
0.5(0.1)
0.09(0.03)
0.4(0.1)
0.07(0.02)
0.08(0.04)
0.02(0.01)
0.19(0.05)
0.40 (0.06)
19.7(4.2)
16.2(3.5)
11.9(1.4)
15.4(3.2)
10.1(1.0)
30.6(6.3)
11.8(3.9)
40.5 (8.0)
1.21 (0.40)
4.85 (0.67)
1.97(0.38)
2.55(0.29)
3.16(0.50)
3.13(0.51)
1.26(0.30)
1.20(0.22)
16.6(4.0)
14.3(3.1)
9.9 (0.8)
11.9(2.8)
7.1(0.8)
26.4 (6.0)
8.9(3.2)
34.8(7.6)
0.87(0.26)
2.60(0.41)
1.20(0.25)
1.01 (0.08)
1.56(0.39)
2.40 (0.4)
0.55(0.16)
0.37(0.08)
3.1(1.7)
1.9(0.4)
2.0(0.8)
3.5(0.5)
2.6(0.2)
4.1 (0.6)
3.5(1.0)
5.7(0.6)
0.34(0.10)
2.25 (0.54)
0.78(0.23)
1.46(0.22)
1.08(0.20)
0.72(0.12)
0.71 (0.18)
0.83(0.25)
0.22(0.05)
0.14(0.01)
0.20 (0.08)
0.32(0.03)
0.37(0.01)
0.17(0.04)
0.41 (0.12)
0.18(0.04)
0.40 (0.06)
1.05(0.32)
0.80(0.31)
1.30(0.12)
0.59(0.18)
0.31 (0.03)
1.65(0.51)
3.38(1.59)
° Standard error in parentheses.
t phosphorus responses 501
V
ASE
•
ASS
0
•
X
50
20
30
40
Total dry weight of control P plant (g)
AT
BD
FF
0
FP
+
FT
A
V
PL
s
PV
PMIO
60
Fig. 1. The total masses of plants grown under P stress (Low P) and control P (High P) are compared for each species. (Species abbreviations can
be found in Table 1.) Each point represents the high P and low P values for a replicate pair within each species. C 3 species data are surrounded by
the thin line while the C 4 species data are surrounded by the thick line. C 3 -C 4 species fall within both areas. Data lying in the lower right corner
of the figure indicate that these species perform well with sufficient P, but poorly with limited P and can be called P-responsive; species less
responsive to P lie in the upper left corner.
Table 3. Average values of plant dry weights, grouped by photosynthetic
type (C3, CA or Cy-C^ intermediate), from P stressed
P treatment) plants and control P plants (High P treatment) grown for 7.5 weeks in a P-buffered sand culture system"
(Low
See Materials and methods section above for details about the statistical model. Values within the same column are not statistically different at P =
0.05 unless noted by lower case letters; values in the same column with identical superscripts are not different. Flower dry weights and branches/tillers
are not shown since not all species flowered and/or branched at low P.
Dry weight per plant (g)
Total
High P
c 3 -c 4
Shoot
Low P
22 2(4.5)b
1.8(0.2)
10.7(2.4)a
2.5(0.4)
11.8 (2.1)ab 1.6(0.3)
HighP
Root shoot
Root
Low P
18.7(4.0)b 1.0(0.2)
8.9(2.2)a
1.4(0.2)
10.5 (1.5)ab 0.8(0.2)
HighP
Low P
HighP
Low P
3.5(O.5)b
1.8(0.5)a
2.5(0.6)ab
0.79(0.11)
0.98(0.23)
0.71(0.14)
0.25(0.03)
0.24(0.03)
0.30(0.08)
1.6(0.3)
0.6(0.1)
1.2(0.3)
' Standard error in parentheses.
Table 4. Values for photosynthetic phosphorus use efficiency and its components, CER and P area ', of P deficient (Low P treatment)
and control P (High P treatment) plants grown in a P-buffered medium"
CER (^mol m-'s-)
P area ' (gm~ 2 )
PPUE
^moi s
Alloteropsis semialata eikloniana
Alloteropsis semialata semialata
Amaranthus tricolor
Brachiaria dictyoneura
Flaveria flondana
Flaveria pringlei
Flaveria trinerva
Panicum laxum
Panicum milioides
Phaseolus vulgaris
" Standard error in parentheses.
HighP
Low P
High P
Low P
Low P
17.0(7.4)
21.1 (3.8)
25.0(3.5)
29.4(9.5)
37.7(2.8)
25.9(3.3)
5.4(2.9)
20.3(3.3)
32.8 (8.0)
21.8(3.0)
15.0(3.1)
13.5(2.6)
22.9(4.2)
19.4(1.3)
22.4(2.9)
14.2(1.3)
7.7(3.9)
11.3(2.3)
17.7(3.7)
11.9(2.5)
0.310(0.126)
0.333(0.129)
0.283 (0.060)
0 302 (0.066)
0.295(0.043)
0.219(0.057)
0.188
0.193(0.018)
0.185(0.073)
0.157(0.010)
0.066(0.011)
0.124(0.028)
0.143(0.015)
0.055 (0.006)
0.149(0.017)
0.056 (0.004)
0.138(0.019)
0.063 (0.009)
0.095(0.019)
0.056 (0.007)
273(68)
144(27)
182(44)
374 (40)
154(16)
260(23)
54 (24)
193(42)
236(45)
209(36)
g
)
502
Halsted and Lynch
•
LowP
YX HighP
0.15
0.1 -
0.05 -
C3 C4
Int
Photosynthetic Type
C3 C4
Int
Photosynthetic Type
C3 C4
Int
Photosynthetic Type
Fig. 2. The average values of PPUE, CER and P area ' for the different photosynthetic types under stressful P (Low P) or control P (High P)
conditions. Measurements were taken ~ 7 weeks after transplanting. T-bars represent ± 1 s.e. Identical letters above the bars within a graph indicate
that values are not significantly different at P = 0.05.
Table 5. Resulting plant dry weight and PPUE means when
Whereas similar growth responses within genera were
species data are grouped into monocot and dicot classes
observed, PPUE varied among species of the same genus.
For example, the PPUE of Alloteropsis eikloniana, the C3 Values are per plant. Plants were grown under both stress (Low P) and
control (High P) P conditions for 7.5 weeks in a P-buffered sand
ecotype, was greater than that of the analogous C4
culture system. Standard errors are in parentheses.
ecotype (Table 4). Likewise, the C3 dicot, F. pringlei, had
a higher PPUE than the analogous C4 species, F. trinervia.
Dicot
P level
Monocot
However, the intermediate Panicum species, P. milioides,
Total dry weight (g)
High
12.5(3.0)
18.8(3.0)
had a higher PPUE than the analogous C3, P. laxum.
Low
2.0(0.2)
2.0(0.3)
Differences in PPUE among closely related species was
Shoot dry weight (g)
High
11.0(2.8)
15.4(28)
Low
1.2(0.2)
1.0(0.1)
due to variability in leaf P content rather than CER
High
3.3(0.5)
Root dry weight (g)
2 0(0.4)
(Fig. 2).
Low
0.77(0.16)
0.91 (0.11)
Though the main focus was concerned primarily with
Root shoot"'
High
0.25(0.04)
0.27(0.03)
Low
0.9(0.2)
12(0.3)
the PPUE of plants under P stress it is important to note
2
C£7?(/xmolm~ s~')
High
23 5(3.0)
26.3(2.9)
the effect of P level on both leaf P content and CER.
Low
14.8(1.3)
17.6(1.6)
Compared to non-stressed plants, P-stressed plants had
P area" 1 (g m~2)
Low
0.078(0.008)
0.11 (0.01)
188(17)
251 (28)
lower leaf P content and lower CER (Table 4; Fig. 2). At />/>[/£• (/xmolg-' s"1) Low
high P, C 3 and C4 average CERs were 21 ^mol m" 2 s" 1
while C 3 -C 4 CER averaged 35 /*mol m"2 s"1. At low P,
Though P area ' was lower in monocots (Table 5), the
C 3 -C 4 CER was significantly reduced and slightly higher
leaf area, leaf number and hence, leaf dry weight, of low
than C3 CER.
P plants were closer to high P values in monocots than
in dicots.
Differences between monocot and dicot species
The most prominent difference in root growth was
between monocots and dicots. Lateral roots of monocots
Though not an initial objective of this study, significant
were much thinner than the lateral roots of dicots. In a
differences were observed between monocots and dicots
later experiment this observation was quantified.
in their response to P stress. Total mass was greater in
Presented here are data from Zea mays (C4 monocot)
dicots at high P, but equivalent between the two classes
and Phaseolus vulgaris (C3 dicot) grown in low P condiat low P (Table 5). The monocot species had lower mean
1
tions (Table 6). The bulk specific root length (i.e. metre
P area" (Table 5) than dicot species, as well as slightly
of root per g of root dry weight) was shorter in monocots
lower mean CER (Table 5); as a result, the average
2
1
1
than dicots (Table 6), apparently because the main root
monocot PPUE was 250 ^mol m' s" g" P, about 50
was
thicker in monocots than dicots. Though data from
units higher than average dicot PPUE.
only
two species are presented here this phenomenon was
The sensitivities of leaf number, leaf area and leaf dry
observed
on many species in all experiments.
weight to P stress were more dependent on class (i.e.
monocot versus dicot) than species (Fig. 3). Under P
stress, leaf production was less reduced in monocots than
Discussion
in dicots. This trend was observed across all photosynGrowth responses to P stress
thetic types; when C3 monocots were compared to C3
dicots, the monocots had higher percentage control
There was a great deal of genetic variability among the
values. This was true for C4 and C 3 -C 4 species as well.
species tested in these studies; however, all species grew
C3/C4 phosphorus responses
leaf dry wt
Dicot
leaf area
Monocot
Class
Fig. 3. Leaf number, leaf area and leaf dry weight of P deficient plants
grown in a P-buffered, soilless medium represented as a percentage of
values from high P plants. Data are grouped by classes. Vertical lines
on bars are ±s.e.; n = 35-39 for monocots; n = 34 for dicots.
Table 6. Average specific root length and main root width of
Phaseolus vulgaris and Zea mays under P stress
Standard error is in parentheses, « = 5 for/' vulgaris, n = 4 for Z. mays.
P. vulgaris
Z mays
Specific root length
(rag" 1 )
Width of main root
(mm)
415(37)
223 (33)
1.12(0.21)
2.20(0.18)
less under P stress and were characterized by growth
patterns previously described for other species under P
stress. Leaf area reductions were observed (Lynch el al,
1991; Qui and Israel, 1992). Leaf numbers as well as leaf
expansion decreased under P stress (Lynch el al, 1991;
Christie and Moorby, 1975; Radin and Eidenbock, 1984).
Low P decreased shoot branching in beans (Lynch et al,
1991) and reduced tiller number in grasses (Christie and
Moorby, 1975). An increase in root-to-shoot ratio was
often observed (Smith et al., 1990; Lynch et al, 1991).
Phosphorus stress has also been reported to decrease
carbon fixation (Fredeen et al, 1990; Usuda and
Shimogawara, 1991) as observed in this study; however,
as in other long-term studies (Sanoeka et al, 1989; Lynch
et al, 1991; Qui and Israel, 1992), CER was not altered
to the same extent as growth. Studies have suggested that
P stress can result in decreased CO 2 fixation and assimilation (Abadia et al, 1987; Rao and Terry, 1989; Sawada
et al, 1983). Suggested mechanisms include the limitation
of the triose-phosphate translocator function (Heber and
Heldt, 1981), limitations of P ; recycling (Stitt, 1986) and
a decrease in ribulose bisphosphate (RuBP) regeneration
(Abadia et al, 1987).
503
Photosynthetic phosphorus use efficiency
Photosynthetic phosphorus use efficiency (PPUE) was
not significantly different between C 3 and C 4 plants in
this study. The hypothesis had predicted that C 4 plants
would have relatively higher PPUE under P stress. It was
thought that during P stress, the anatomical and biochemical compartmentation of the C 4 carbon fixing system
would allow leaves to concentrate P, in bundle sheath
cells, the cells responsible for most carbon assimilation,
rather than require them to distribute it throughout the
mesophyll and bundle sheath cells. Concentration of Pi
in the bundle sheaths would assist the phosphate/triosephosphate translocators of the chloroplasts maintain
function (export of assimilates out of the chloroplasts
and import of P ; into the chloroplasts) during P stress.
As a result, C 4 CER would require less P, and be less
sensitive to P stress than C 3 CER.
As predicted, CERs of C 4 plants under P stress were
less reduced than C 3 CERs; however, C 4 species had
higher leaf P concentration per unit leaf area than
expected. Among species within a genus the C 4 species
had higher leaf P content than either the C 3 or C 3 -C 4
intermediate species. These contrasting leaf P levels
resulted in more discernible PPUE differences between
the C 3 and C 4 species in a genus, with the C 4 being lower
than the C 3 . Although concentration of P; in the bundle
sheath cells of C 4 plants can not be ruled out, if such a
phenomenon does exist, it does not result in either lower
total P area" 1 in leaves or higher photosynthetic phosphorus use efficiency as defined here.
Photosynthetic phosphorus use efficiency is an instantaneous parameter, whereas growth integrates stress tolerance over time. It was observed that photosynthetic
phosphorus use efficiency was not highly correlated with
growth or sensitivity to P stress. Though C 4 species in
general had a slightly lower PPUE than C 3 species, P
stress did not limit their growth any more than the growth
of C 3 species; the average plant biomass under P stress
was slightly higher in the C 4 species than in either the C 3
or C 3 -C 4 intermediate species. Figure 1 shows that the
C 4 plants were less sensitive to P stress, i.e. the total
biomass production was less changed by P stress in C 4
species than in C 3 . Intermediate species had intermediate
sensitivity. The poor correlation of PPUE with growth
under P stress supports the suggestion that C allocation
and utilization are more important than instantaneous C
assimilation in determining the growth of plants under P
stress (Lynch and Beebe, 1994).
Species differences
The P stress tolerance of the species varied widely. The
biomass production of crop species, Amaranthus tricolor,
Phaseolus vulgaris, and Zea mays was most sensitive to P
stress while the wild Flaveria and Panicum species were
504
Halsted and Lynch
intermediate in sensitivity. Brachiaria dictyoneura not only
produced the highest biomass under P stress, but also
had the lowest sensitivity, as well as extremely low per
cent P levels in both root and leaf tissues under P
deficiency (Halsted, 1994). It also increased its root-toshoot ratio under P stress, as did many other species, but
its P distribution patterns remained similar to those
observed for high P. The combination of these three
things: tolerance to low tissue P levels, increased root-toshoot ratio and an unchanged root P to shoot P ratio
under P stress, conferred a stress tolerance not seen in
any other species in the study.
Differences between monocots and dicots
Both monocot C3, C4 and C 3 -C 4 species and dicot C3,
C4 and C3-C4 species were included in this study so that
a range of responses to P stress could be observed. No
consistent growth differences between C3 and C4 types
were observed. Though not originally intended as a
comparative study of monocots and dicots, these experiments did reveal consistent differences both in the sensitivity of leaf production to low P and root morphology
between monocots and dicots. These observations lead
us to speculate on the role of taxonomic class in explaining
one of the observations that led to this study: in tropical
legume-based pasture systems, grasses, many of them C4,
aggressively displace C3, dicot legumes. Whereas differences in PPUE and photosynthetic type were anticipated,
in explaining this observation perhaps taxonomic class is
more relevant.
The superior growth of monocot leaves under P stress
may be due to the localization of leaf growth in the basal
meristem, as opposed to the diffuse expansive growth of
dicot leaves. Since only a small part of the lamina
elongates at any one time, perhaps a lower amount of P
coming to a leaf during P stress has less impact on growth
than it does in dicots, where P is needed throughout the
young, growing leaf. Another possible explanation is
suggested by the observation that monocot leaves under
N stress expand more than dicot leaves under equivalent
N stress (Radin, 1983). Radin argued that this is due to
the reduced hydraulic conductivity in N-deficient plants:
monocot leaves continue expansion because their growth
zone is protected from dehydration by the sheath of the
subtending leaf, whereas midday water deficit in dicot
leaves reduces leaf expansion. Although this observation
was not repeated with P stress, later work showed that P
deficiency is also linked to reduced hydraulic conductivity
(Radin and Eidenbock, 1984), suggesting that the same
mechanism may apply. The validity of these two hypotheses could be worked out by careful monitoring of
water potential, P content, and expansion in the growth
zone of monocot and dicot leaves under varying P
regimes. The different leaf sensitivities observed here did
not result in differences in total biomass production in
this study, but may have some bearing on competitive
interactions in the field. The finer lateral roots of monocots initially suggests that they would be able to explore
a volume of soil in 'search' of P with lower carbon costs
than those incurred by dicot roots. Further research
elaborating on root system architecture would clarify
this issue.
Conclusions
In this study C3, C4 and C 3 -C 4 intermediate species were
surveyed for differences in PPUE and growth responses
to realistic, low P conditions. Significant differences in
PPUE between C3 and C4 species were not observed,
though greater differences can be observed among species
within a genus. While CER was decreased by P stress as
expected, P area"1 was more reduced in C3 than in C4
taxa, contrary to the hypothesis. Monocots had lower
leaf P content and better maintenance of leaf production
under P stress than dicots. Further research on the
particularly tolerant species, B. dictyoneura, including P
localization within leaf tissue types (mesophyll and bundle
sheath cells) will provide relevant information on plant
response and tolerance to P stress.
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