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0 Department of Ecology and Evolution, State University of New York, Stony Brook, New York 11794 USA
Received for publication August 17, 1998. Accepted for publication June 29, 1999.
ABSTRACT
Theoretical considerations have suggested that there may be differences in photosynthetic nitrogen use efficiency (PNUE) among plants that use different biochemical variants of C4 photosynthesis. To test this hypothesis we examined the leaf nitrogen content and photosynthetic rates of six grass species (three of C4 subtype NAD-ME and three of C4 subtype NADP-ME) grown over a wide range of nitrogen supply. While there were significant differences among the species in various traits, there were no consistent differences between the C4 subtypes in either leaf nitrogen content at a given level of nitrogen supply or in the leaf nitrogen-photosynthesis relationship. We suggest that species-level variation in photosynthetic nitrogen use efficiency among C4 species is large enough to mask any differences that may be due to C4 subtype.
Key Words: C4 photosynthesis • grasses • leaf nitrogen • NAD-ME photosynthesis • NADP-ME photosynthesis • nitrogen use efficiency • photosynthetic nitrogen use efficiency • Poaceae
Numerous studies have identified differences between C3 and C4 plants in nitrogen usage and photosynthetic nitrogen use efficiency (PNUE; photosynthesis per unit leaf nitrogen), which result from their different modes of carbon fixation (Brown, 1978
; Bolton and Brown, 1980
; Sage and Pearcy, 1987
). The ability of C4 plants to concentrate CO2 at the sites of carboxylation allows them to attain higher photosynthetic rates for a given content of RUBISCO (ribulose bisphosphate carboxylase) than C3 plants, resulting in a higher photosynthetic nitrogen use efficiency (Ehleringer and Monson, 1993
). The mechanism of C4 photosynthesis, however, is not uniform among C4 plants; there are three distinct biochemical pathways of C4 photosynthesis, each performed by different C4 species (Hatch, 1987
). Previous research has suggested that the biochemical differences among these C4 variants have implications for their nitrogen usage (Bowman, 1991
), but very little work has been done comparing nitrogen use among C4 species that use different C4 pathways.
The three different pathways of C4 photosynthesis differ in the mechanisms used to transport CO2 to bundle sheath cells and to regenerate phosphoenol pyruvate (PEP). They are termed the NAD-ME, NADP-ME, and PCK pathways, after the principal bundle sheath decarboxylation enzyme used in each (NAD malic enzyme, NADP malic enzyme, and phosphoenolpyruvate carboxykinase, respectively; Hatch, 1987
). Of these, the NAD-ME and NADP-ME are the most common and have the widest taxonomic distribution, and we will confine our discussion to these two types.
Bowman (1991)
points out that several of the differences between these pathways might have implications for their efficiency of nitrogen use. He notes that the NAD-ME pathway is more complex than the NADP-ME, involving a greater number of transformations of intermediates. This biochemical complexity in NAD-ME species imposes an extra nitrogen cost for the construction of the enzymes that mediate each reaction. In addition, the NAD-ME pathway requires a pool of nitrogen for the amination of OAA to aspartate, and of pyruvate to alanine. On the basis of these differences in carbon metabolism, Bowman (1991)
hypothesized that NADP-ME species would have a higher rate of photosynthesis than NAD-ME species at a given leaf nitrogen concentration. If this is the case, NAD-ME species must either have lower photosynthetic rates than NADP-ME species, or they must have higher nitrogen concentrations in their photosynthetic organs than NADP-ME species.
There is some empirical evidence that NAD-ME and NADP-ME species differ in the nitrogen concentration of their photosynthetic tissues. Bowman (1991)
, comparing four NAD-ME and two NADP-ME members of the grass genus Panicum, found that at high levels of nitrogen availability the NAD-ME species had higher shoot nitrogen concentrations than the NADP-ME species. LeCain and Morgan (1998)
found that leaf nitrogen concentration was higher in three NAD-ME grass species than in three NADP-ME grasses. These findings suggest that NAD-ME species may compensate for a lower PNUE with greater allocation of nitrogen to photosynthetic organs. However, no previous study has compared the relationship between leaf nitrogen concentration and photosynthesis among C4 subtypes to see whether the biochemical differences among the pathways translate into the expected differences in leaf-level physiological performance.
A lower photosynthetic return on nitrogen allocated to leaves for NAD-ME than NADP-ME photosynthesis might have consequences for the distribution of species that use the different C4 subtypes across natural environmental gradients of nitrogen. Pons, Van der Werf, and Lambers (1994)
found that C3 monocot species with a low PNUE have low potential growth rates (the growth rate achievable under ideal growing conditions). Ecologically, low potential growth rates are associated with species found in nitrogen-poor habitats (Lambers and Poorter, 1992
), and a low PNUE for NAD-ME species might similarly limit them to low-fertility habitats.
To explore possible differences in the photosynthetic response of C4 subtypes to nitrogen, we compared the relationship between leaf nitrogen content and photosynthetic rate for three species of NAD-ME and three species of NADP-ME grass, grown over a wide range of nitrogen supply. In addition to the photosynthesis-leaf nitrogen relationship, we also compared the relationship between nitrogen availability (fertilizer N concentration) and leaf nitrogen concentration to test the robustness of previous research, which found higher nitrogen concentration in the photosynthetic organs of NAD-ME species.
MATERIALS AND METHODS
Experimental procedures
Seeds for the experiment were collected from populations in Suffolk County, New York, USA in the autumn of 1994. Collections were made from similarly situated sites within an area of ~2 km2 to minimize differences among the populations in adaptations to climate. The six co-occurring species chosen were Eleusine indica, Eragrostis cilianensis, and Panicum virgatum with the NAD-ME subtype and Schizachyrium scoparium, Digitaria ischaemum, and Setaria glauca with the NADP-ME subtype (Hattersley, 1987
; Hattersley and Watson, 1992
).
Seeds were germinated in flats of sand and transferred to plastic pots containing 600 mL of washed sand (one plant per pot). Sand particle size was predominantly between 500 and 1400 µm. Plants were grown under natural light from 3 May to 7 August 1995 in the Life Sciences greenhouse at the State University of New York in Stony Brook, New York, USA (40°55'20'' N, 73°07'50'' W). Maximum photosynthetic photon flux density was ~1200 µmol photons·m-2·s-1. Mean daily minimum and maximum temperatures were 20.8°C (SD = 3.3) and 33.0°C (SD = 4.2), respectively. Pots were watered to excess daily with a nutrient solution containing 2 mmol/L K2PO4, 1 mmol/L CaSO4, 2 mmol/L MgSO4, 40 µmol/L FeNa EDTA, 50 µmol/L KCl, 33 µmol/L H3BO4, 2 µmol/L MnSO4, 2 µmol/L ZnSO4, 0.5 µmol/L CuSO4, and 0.5 µmol/L MoO3. Nitrogen supply was varied as NH4NO3 at concentrations of 0.01, 0.05, 0.1, 0.5, 1, 10, 20, and 40 mmol/L.
Gas exchange measurements were made with an LI-6400 gas exchange system (LI-COR, Lincoln, Nebraska, USA). Measurements were made on the newest fully expanded leaf on plants 7–14 wk after seedling emergence. One leaf was measured per plant. Between 16 and 18 plants were measured for each species. Light was supplied by the LI-6400's LED light source at a photosynthetic photon flux density of 1500 µmol photon·m-2·s-1 (maximum irradiance at 670 nm). Air entering the leaf chamber was drawn from a buffered volume of ambient greenhouse air (occasionally the air stream was dried partially over desiccant to avoid condensation in the system). Intake CO2 levels were ~370 ppm. After the leaf had acclimated to the chamber, as indicated by steady photosynthetic rates, measurements were made at 30°C, following which the temperature was raised to 37°C, the leaf allowed to re-acclimate, and photosynthesis and transpiration were measured again. Leaf temperature was controlled by use of the LI-6400's Peltier thermo-electric heat exchangers and a hot-air gun. Measurements were made with leaf temperatures held within 0.5° of the target. Net photosynthetic rate (Ps) was calculated as per LI-COR (1995) by the LI-6400 software (Open 1.0). Immediately following the gas exchange measurements, leaf area was recorded with an LI-3000A area meter (LI-COR, Lincoln, Nebraska, USA) and the leaf was excised from the plant and dried at 65°C. Leaf nitrogen concentration was determined using a Carlo Erba (Milan, Italy) EA 1108 CHNS analyzer at the Analytical Laboratory Facility of the Marine Sciences Research Center, State University of New York at Stony Brook.
PNUE
There are a variety of ways to calculate PNUE, of which the simplest and most commonly used is the ratio of photosynthetic rate to leaf nitrogen concentration. An alternative approach, followed here, is to derive PNUE from the regression of photosynthesis on leaf nitrogen; PNUE is indicated by the height of the regression line at a given nitrogen concentration. This approach facilitates both comparison of PNUE at any given leaf nitrogen concentration and examination of how PNUE varies with leaf nitrogen (e.g., comparison of regression slopes). When discussing PNUE differences in our own results, it is in this sense, as indicating differences in the relative height of this regression; in discussing the results of others we assume the definition of PNUE used by the original authors of these studies.
Statistical analyses
In this study, our interest was primarily in potential differences among species and C4 subtypes in the relationships between pairs of measured variables. These included the relationships of leaf nitrogen concentration with photosynthetic rate and of fertilizer nitrogen with leaf nitrogen concentration. To test the significance of differences among the species and between the C4 subtypes we examined these relationships with separate analysis of covariance (ANCOVA) models. For each model, the first variable of a pair listed above (e.g., leaf nitrogen concentration) was included as a covariate with the second (e.g., photosynthetic rate) as the dependent variable and C4 subtype as a categorical factor. Each model also included species as a factor nested within C4 subtype. As the covariates used in these analyses were measured with error, a Model II approach would have been strictly correct. However, in the absence of a well-developed Model II ANCOVA methodology (Sokal and Rohlf, 1995
) we followed general custom (Goldberg and Scheiner, 1993
) and used a Model I approach. Each regression was based on measurements for 16–18 plants.
Recent studies have suggested that the relationship between leaf nitrogen concentration and photosynthetic rate captures different aspects of the functional biology of leaves when these variables are expressed on a leaf area vs. a leaf mass basis (Reich and Walters, 1994
; Reich, Walters, and Ellsworth, 1992
). We therefore present data on both bases for comparison.
Leaf nitrogen concentration, photosynthetic rate, and nitrogen fertilizer concentration were all log transformed prior to significance testing to better meet the assumptions of the statistical tests used. These transformations led both to more normal distributions for these variables and to better linear fits between pairs of variables. Statistical analyses were performed using the linear model procedures (Fit Model routine) of the JMP 3.0.2 package for the Macintosh (SAS Institute, Cary, North Carolina, USA).
RESULTS AND DISCUSSION
The rate of photosynthesis obtained for a given leaf nitrogen concentration on a leaf-mass basis differed substantially among the species used in this study (Figs. 1, 2). With the exception of Schizachyrium, there is a clear ranking of the species in this regard across the entire observed range of leaf nitrogen concentrations (i.e., the regression lines do not cross), with Eragrostis > Digitaria > Setaria > Panicum > Eleusine (Fig. 2). There is no clear pattern of difference between the NAD-ME and NADP-ME species. Two of the NAD-ME species, Panicum and Eleusine had the lowest PNUE of the six species, as hypothesized by Bowman (1991)
. However, the third NAD-ME species, Eragrostis had the highest rate of photosynthesis of any of the species at any given leaf nitrogen concentration.
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Very similar patterns were found when leaf nitrogen and photosynthetic rate were expressed on a leaf area rather than leaf mass basis. The rankings of the species in PNUE were similar to those found on a leaf mass basis, with Eragrostis again having the highest, and Eleusine the lowest PNUE of the species (Figs. 3, 4). There were statistically significant differences among the species in photosynthetic rates at a given leaf nitrogen concentration on a leaf-area basis (Figs. 3 and 4 for 30°C; data for 37°C not shown as relationships are essentially identical to those at 30°C; df = 4,80; F = 9.8; P < 0.001 at 30°C; df = 4,69; F = 10.31; P < 0.001 at 37°C). However, the NAD-ME and NADP-ME species did not differ significantly in either the slope (df = 1,4; F = 0.14; P > 0.50 at 30°C; df = 1,4; F = 0.02; P > 0.75 at 37°C) or intercept (df = 1,4; F = 0.56; P > 0.25 at 30°C; df = 1,4; F = 0.002; P > 0.75 at 37°C) of the leaf nitrogen-photosynthesis relationship at either 30°C or 37°C (Fig. 4 for 30°C; data for 37°C omitted for brevity, as relationships are substantially the same as those at 30°C).
Leaf nitrogen concentration shows a similar pattern. There are substantial differences among the species in leaf nitrogen concentration at a given level of nitrogen availability, whether leaf nitrogen was expressed on a per-leaf mass (Figs. 5, 6), or a per-leaf area basis (Figs. 7, 8). There are differences in the rankings among the six species in leaf nitrogen concentration depending on which basis of measurement is used. On a per-mass basis, the highest leaf nitrogen concentrations over most of the range of fertilizer nitrogen used here were for two of the NAD-ME species, Eleusine and Eragrostis. However, the third NAD-ME species, Panicum, had leaf nitrogen concentrations comparable to the NADP-ME species. On a leaf area basis, Eleusine had the highest leaf nitrogen concentration of any of the species, and at any level of fertilizer nitrogen, three out of the four species with the highest leaf nitrogen were NAD-ME (Fig. 8). These results are consistent with previous observations of higher leaf and shoot nitrogen concentrations in NAD-ME than in NADP-ME grasses (Bowman, 1991
; LeCain and Morgan, 1998
).
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These results point to a pattern of substantial variation in PNUE and in leaf nitrogen concentration at the level of the species. Of the three NAD-ME species in this study, only Eleusine conformed to the predicted patterns of a low PNUE and a high leaf nitrogen concentration. Eragrostis had high leaf nitrogen concentration, but also had the highest PNUE of any species in the study. Panicum, on the other hand, combined low PNUE with a moderate leaf nitrogen concentration. A relatively low nitrogen concentration (for an NAD-ME species) appears to be a consistent attribute of Panicum virgatum. Both Bowman (1991)
and LeCain and Morgan (1998)
found that P. virgatum had the lowest shoot or leaf nitrogen of the NAD-ME species in their studies.
While we do not know the causes of the interspecific variation observed, some possibilities can be suggested by reference to other studies that have compared the use of nitrogen for photosynthetic machinery among a number of species (Evans, 1989
; Usuda, Ku, and Edwards, 1984
). One possible explanation for the differences among the species in photosynthetic rates at a given level of leaf nitrogen is that it is due to differences in the proportion of leaf nitrogen that is used for photosynthetic enzymes rather than for other leaf constituents (structural proteins, chlorophyll, nucleic acids, etc.). This has been proposed as a partial explanation for differences in photosynthetic nitrogen use efficiency among C3 plants (Evans, 1989
; Pons, Van der Werf, and Lambers, 1994
), and such variation may be expected to occur among C4 species as well. The species may also differ in their leaf concentrations of inorganic nitrogen.
Future experiments that distinguish among leaf nitrogen partitioned to these various compartments may be sensitive to differences among the C4 subtypes that are missed with the whole-leaf nitrogen approach followed here. For example, there may be differences between the NAD-ME and NADP-ME subtypes in photosynthetic rates per-leaf organic nitrogen or per-leaf soluble protein even in the absence of differences in PNUE on a total nitrogen basis.
This study, having sampled only three species each of the NAD-ME and NADP-ME C4 subtypes, also cannot rule out the possibility that there might exist small but consistent differences between NAD-ME and NADP-ME species in leaf total nitrogen concentration or PNUE, as suggested by Bowman (1991)
. Indeed, while we could detect no consistent differences between the C4 subtypes, Bowman (1991)
and LeCain and Morgan (1998) found NAD-ME species to have higher nitrogen concentrations than NADP-ME species in shoots and leaves, respectively, and we detected a trend in the same direction. However, there appears to be substantial overlap in PNUE between NAD-ME and NADP-ME species.
This result stands in stark contrast with the comparison of PNUE in C3 vs. C4 species. The difference in photosynthetic nitrogen use efficiency on a total leaf nitrogen basis between these photosynthetic types is large and has been consistently found, even in studies comparing unrelated C3 and C4 species (Bolton and Brown, 1980
; Sage and Pearcy, 1987
). The present study shows that any differences in photosynthetic nitrogen use efficiency between the C4 subtypes must be small and any such differences between the C4 subtypes occur against the background of substantial interspecific variation in PNUE.
FOOTNOTES
1 The authors thank Christiane Biermann, Rich Fallon, Proserpina Gomez, and Amal Jasentuliyana for help with planting and maintaining the plants; Linda D'Anna for assisting with the gas exchange measurements; David Hirschberg for performing the elemental analysis of the leaves; Naomi Dicker for helping with identification of the plant species; Valerie Tierce for allowing us use of a hot air gun; Mike Axelrod and John Klumpp for multifaceted assistance; and Zoe Cardon, Jessica Gurevitch, Charles Janson, Deborah Goldberg, Russell Monson, and several anonymous reviewers for comments on drafts of this paper. This work was supported by a Sigma Xi grant to DRT. This is contribution 1046 in Ecology and Evolution, State University of New York at Stony Brook. 
2 Current address: Biological Sciences Center, Desert Research Institute, 2215 Raggio Parkway, Reno, Nevada 89512 USA.
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Pons, T. L., A. Van der Werf, and H. Lambers. 1994 Photosynthetic nitrogen use efficiency of inherently slow- and fast-growing species: possible explanations for observed differences. In J. Roy and E. Garnier [eds.], A whole plant perspective on carbon-nitrogen interactions, 61–77. SPB Academic, The Hague, The Netherlands.
Reich, P. B., and M. B. Walters. 1994 Photosynthesis-nitrogen relations in Amazonian tree species. II. variation in nitrogen vis-a-vis specific leaf area influences mass- and area-based expressions. Oecologia 97: 73–81. [CrossRef][ISI]
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Sokal, R. R., and F. J. Rohlf. 1995 Biometry. W. H. Freeman, New York, New York, USA.
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