269 Harrison etal Nitrogen in cell walls (final PCE08-149) 3

1Nitrogen in cell walls of sclerophyllous leaves accounts for little of the 2variation in photosynthetic nitrogen use efficiency 4Matthew T Harrison1,2, Everard J Edwards1,*, Graham D Farquhar1, Adrienne B 5Nicotra2 and John R Evans1 71) Environmental Biology Group, Research School of Biological Sciences, The 8Australian National University, GPO Box 475, Canberra ACT 2601, Australia 92) School of Botany and Zoology, The Australian National University 10Canberra, ACT 0200, Australia 11* Present address: CSIRO Plant Industry, Private Mail Bag, Merbein, VIC 3500, 12Australia 13 1 1ABSTRACT 2Photosynthetic rate per unit nitrogen generally declines as leaf mass per unit area 3(LMA) increases To determine how much of this decline was associated with 4allocating a greater proportion of leaf nitrogen into cell wall material, we compared 5two groups of plants The first group consisted of two species from each of eight 6genera; all of which were perennial evergreens growing in the Australian National 7Botanic Gardens The second group consisted of seven Eucalyptus species growing in 8a greenhouse The percentage of leaf biomass in cell walls was independent of 9variation in LMA within any genus, but varied from 25 to 65% between genera The 10nitrogen concentration of cell wall material was 0.4 times leaf nitrogen concentration 11for all species apart from Eucalyptus, which was 0.6 times leaf nitrogen concentration 12Between 10 and 30% of leaf nitrogen was recovered in the cell wall fraction, but this 13was independent of LMA No trade-off was observed between nitrogen associated 14with cell walls and the nitrogen allocated to Rubisco Variation in photosynthetic rate 15per unit nitrogen could not be explained by variation in cell wall nitrogen 16 17Keywords 18Cell wall nitrogen, leaf mass per unit area, nitrogen allocation, photosynthetic 19nitrogen-use efficiency, Rubisco, structural nitrogen 20 21 1INTRODUCTION 2The photosynthetic capacity of a leaf is generally well correlated with leaf nitrogen 3content Although this relationship varies between species, much of the variation is 4related to another leaf parameter, specific leaf area (SLA), the projected leaf area per 5unit leaf dry mass Thus there exists a global function, regardless of life-form or 6location, which can predict photosynthetic capacity per unit leaf dry mass from 7nitrogen concentration and specific leaf area (Reich et al., 1997, Wright et al., 2004) 8Photosynthetic rate per unit nitrogen (photosynthetic nitrogen use efficiency, PNUE) 9tends to decrease as specific leaf area decreases (Hikosaka, 2004, Poorter & Evans, 101998) Since smaller specific leaf area is associated with greater leaf longevity, Field 11& Mooney (1986) suggested that there may be a trade-off between investing nitrogen 12in photosynthetic proteins such as Rubisco versus compounds required for longevity 13 This hypothesis languished for lack of measurements of structural nitrogen in 14leaves However, Onoda et al (2004) and Takashima et al (2004) developed methods 15for extracting detergent soluble proteins from leaf material They assumed that the 16nitrogen that remained behind represented cell wall protein The comparison between 17evergreen and deciduous Quercus species (Takashima et al., 2004) revealed a clear 18trade-off between nitrogen invested in Rubisco and cell wall proteins Leaves from 19evergreen Quercus had greater leaf mass per unit area (LMA, the reciprocal of SLA) 20and allocated a greater proportion of leaf nitrogen to cell wall protein than leaves from 21deciduous Quercus Leaves of Polygonum cuspidatum also allocated a greater 22proportion of leaf nitrogen to cell walls as leaf mass per unit area increased (Onoda et 23al., 2004) However, for a given leaf mass per unit area, Polygonum allocated a 24smaller proportion of nitrogen to cell walls than Quercus While both of these genera 25provide support for the hypothesis put forward by Field and Mooney (1986), the 1maximum leaf mass per unit area for leaves from both of these studies was only 60 g 2m-2 This is at the lower end of the range reported by Reich et al (1997) and so may 3not be representative of sclerophyllous, long lived leaves Ellsworth et al (2004) analysed leaf photosynthesis from 16 species with leaf 5mass per unit area ranging from 50 to 300 g m-2 They calculated that the proportion 6of nitrogen allocated to Rubisco declined as leaf mass per unit area increased and 7suggested that this was related to the need for greater investment in structural 8nitrogen Clearly there is a need for more data on cell wall nitrogen Therefore, our 9first objective was to sample leaves from species representing a broad range of leaf 10mass per unit area to see whether the proportion of nitrogen allocated to cell walls was 11related to leaf mass per unit area Two sampling strategies were used First, pairs of 12species from each of eight genera growing in the Australian National Botanic Gardens 13were chosen on the basis of contrasting leaf mass per unit area These allowed 14phylogenetically independent contrasts to be made (Felsenstein, 1985) Second, seven 15Eucalyptus species were grown and measured in a greenhouse to enable more 16comprehensive analyses of a single genus over a four-fold range in leaf mass per unit 17area 18 Another feature of the relationship between photosynthetic nitrogen use 19efficiency and leaf mass per unit area is that for a given leaf mass per unit area, 20photosynthetic nitrogen use efficiency varies by an order of magnitude It is likely that 21the nitrogen concentration in leaf structural biomass varies between species and that 22this could account for some of the scatter Therefore, our second objective was to 23assess how much of the variation in photosynthetic nitrogen use efficiency was 24associated with variation in the proportion of leaf nitrogen allocated to cell walls 25 1MATERIALS AND METHODS 2Morphological and physiological measurements were obtained from two independent 3investigations: a field study comparing species pairs from eight genera and a 4greenhouse experiment that examined seven species of the genus Eucalyptus 6Plant material 8A field study was conducted using two C3 species from each of eight perennial 9Australian evergreen genera growing in the Australian National Botanic Gardens 10(ANBG), Canberra (35o 12’’ S 149o 04’’ E) The genera were selected so as to provide 11a wide range of LMA, and thus included a variety of growth forms including vines, 12shrubs and trees (Table 1) 13 The greenhouse study was conducted at the Australian National University, 14Canberra, using twelve month-old seedlings of Eucalyptus bridgesiana, E elata, E 15mannifera, E moorei, E pauciflora, E polyanthemos and E rossii, which were 16purchased from the Yarralumla nursery (Canberra) At the nursery, seedlings were 17grown in potting mix that contained a controlled-release fertilizer (18% nitrogen), 18applied at a rate of kg m-3 All species were grown in full sunlight on the same site 19Seedlings were transplanted from nursery tubes into large plastic pots (180 × 180 × 20240 mm3, length × width × depth) filled with a sterilised sand/peat/perlite mixture on 211/5/2006 22 23Growth conditions and experimental design of the greenhouse study 24 1Five blocks containing 14 eucalypts were arranged in the greenhouse, with each block 2containing two replicates of each species Greenhouses were maintained at 22-25 oC 3during the day and 15-18 oC at night Supplementary lighting (280 µmol 4photosynthetically active radiation (PAR) photons m-2 s-1) was provided by six 150 W 5Crompton PAR38 flood lamps between 0500-1000 and 1700-2100 hrs in order to 6extend day length Midday PAR measured with a quantum light sensor (Li-Cor Inc., 7Lincoln, NE, USA) averaged 500 µmol PAR photons m-2 s-1 on sunny days Seedlings 8were watered to field capacity, twice daily Rorison’s nutrient solution (Hewitt, 1966) 9was applied twice per week to each seedling, 100 mL from May 1st to June 16th and 10625 mL from June 17th to July 1st, to increase the size and growth-rate of young 11leaves For five of the replicates of each species, one per block, the mM Ca(NO3)2 in 12the Rorison’s solution was replaced with mM CaCl2, providing plus- and minus13nitrogen treatments, respectively To distinguish between new and pre-existing leaves, 14white tags were attached to the youngest petiole on the main stem prior to the start of 15the experiment Seedlings were periodically sprayed with chemicals for control of 16psyllids and powdery mildew 17 18Gas exchange measurements 19 20Measurements of CO2 assimilation rate per unit area (Aa) in the ANBG were made at 21saturating irradiance (Table 1), which was determined for each species by first 22measuring a light-response curve Measurements were made on leaves for all species 23except Acacia, where phyllodes were used, from 28/3/2006 to 24/4/2006, using an 24infra-red gas analyser (IRGA) (LI-6400, LI-COR, Lincoln, Nebraska, USA) open gas 25exchange system Where possible, a second LI-6400 was used on adjacent leaves of 1the same plant, allowing cross-checks for consistency in measurement Steady-state 2measurements were made on similar fully expanded young leaves at an ambient CO2 3concentration (Ca) of 375 µmol mol-1, between 0900 and 1500 hrs on sunny days Leaf 4temperature was allowed to follow ambient conditions, which ranged between 15 and 532 ºC 7Photosynthetic light response curves were measured for all seven greenhouse grown 8eucalypt species A standard irradiance of 1800 µmol PAR photons m-2 s-1 was adopted 9for all species during the measurement of CO2 response curves, with the block 10temperature maintained at 22 oC, a flow rate of 500 µmol s-1 and the relative humidity 11of air entering the leaf chamber maintained between 70 and 80% Following 12equilibration, Aa was measured at ambient CO2 (375 μmol mol-1) and a series of nine 13consecutive CO2 concentrations from 50 to 1300 µmol mol-1 As with the ANBG 14measurements, two LI-6400s were used 15 16At the time of measurement, only E elata, E bridgesiana, E mannifera and E 17polyanthemos plants had grown fully expanded, new leaves There were no new 18leaves on minus-nitrogen E polyanthemos plants at this time To allow contrasts 19between all species, at least four pre-existing, non-necrotic leaves on different plants 20from the plus-nitrogen treatment were measured 21 22Scaling photosynthesis to a common Ci value 23 1The model developed by Farquhar, von Caemmerer & Berry (1980) was used to scale 2all Aa measurements to a common intercellular CO2 partial pressure (Ci) of 300 µmol 3mol-1 (Aa300) This approach was carried out using equation (1), assuming the in vivo 4maximum carboxylation activity of Rubisco per unit area (Vcmax) was limiting 5photosynthesis (Aa) Aa = Vcmax ( Ci − Γ∗ ) − Rd Ci + K c ( + O / K o ) (1) 7The kinetic constants of Rubisco (Kc and Ko, the Michaelis-Menten constants for CO2 8and O2 respectively, and Γ*, the CO2 compensation point in the absence of dark 9respiration, Rd) were adopted from von Caemmerer et al (1994) assuming infinite 10internal conductance, with their temperature dependence functions given in von 11Caemmerer (2000) Rd was assumed to be 0.1 of Aa for the ANBG data These 12assumptions were validated from CO2 response curves measured on the eucalypt 13leaves (data not shown) 14 15Morphological measurements 16 17When gas exchange measurements were completed each day, leaves were detached 18and the segment used for measurement of photosynthesis was cut out and weighed 19The segment area was determined using a leaf area meter (Li-Cor L3100, Li-Cor Inc., 20Lincoln, NE, USA) Lamina thickness (T) was measured between the midrib and leaf 21edge with a Mitutoyo analogue thickness gauge (precision ± 20 µm) T was calculated 22as the mean of four measurements All ANBG leaf segments were dried for a 23minimum of 48 h at 80 oC then reweighed, allowing calculation of leaf dry mass per 24unit area (LMA) Water content (WC, mol m-2) was computed as the change in leaf 1mass due to drying, divided by leaf area A duplicate set of leaves, matching those 2used for photosynthesis measurements, was sampled for cell wall nitrogen 3measurement, being snap-frozen in liquid nitrogen, then stored at -80 oC until used 4Leaves were then freeze-dried at -45 oC, 64 mT for at least days, using a 5Microprocessor Controlled Bench-Top Lyophilizer (FTS Systems Inc., Stone Ridge, 6New York, USA) All sampled greenhouse eucalypt leaf segments were freeze-dried 8Leaf nitrogen measurements 10 (a) Total leaf nitrogen 11 12All dried leaves were ground separately in a ball mill The nitrogen concentration of 13the photosynthetic segment was assayed using an elemental analyser (EA 1110 CHN14O Carlo-Erba Instruments, Milan, Italy) with a typical machine precision of ± 0.02% 15N Approximately 1.2 mg of each segment was analysed 16 17 (b) Cell wall mass and nitrogen 18 19A protocol was adapted from Lamport (1965) and Onoda et al (2004) to remove 20soluble protein from the milled leaf material Approximately 10 mg of freeze dried 21leaf was extracted in 1.5 mL of buffer (50 mM Tricine, pH 8.1) containing 1% PVP40 22(average molecular weight 40 000; Sigma Chemical Company, Product No 1407, 23Saint Louis, USA) The sample was vortexed, centrifuged at 12000 g for 24(Eppendorf AG 5424, Hamburg, Germany) and the supernatant carefully removed 25The pellet was resuspended in buffer without PVP containing 1% SDS, incubated at 190 °C for minutes, then centrifuged at 12000 g for This was repeated and 2then two washes with 0.2M KOH, two washes with deionised water and finally two 3washes with ethanol were carried out The tube containing the pellet was then oven 4dried at 80 oC The remaining dry mass of pellet was assumed to represent the leaf 5structural biomass and the N content was determined on 2-5 mg of material using the 6elemental analyser as above 8The fraction of leaf nitrogen in cell wall material, NCW/NL, was calculated using 9equation (2): 10 N CW M CW N CW M L = × × NL M L M CW N L (2) 11where the fraction of cell wall material (MCW) recovered from the total leaf biomass 12(ML) was multiplied by the nitrogen concentration of cell wall material (NCW/MCW) 13divided by the leaf nitrogen concentration (NL/ML) 14 15Attempts were made to extract Rubisco for several of the species using the method 16which has routinely been used for Nicotiana tabacum (Mate et al., 1993) We tried to 17grind fresh leaves, or leaves frozen in liquid N2, using mortar and pestle, a Ten Broeck 18homogeniser, or a Polytron and we also tried to extract freeze dried leaf material that 19was ball milled None of our attempts yielded adequate soluble protein or Rubisco, 20presumably because we were unable to successfully rupture the mesophyll cells 21Calculation of Rubisco nitrogen and photosynthetic nitrogen-use efficiency 22 23The fraction of nitrogen allocated to Rubisco (NR/NL) was calculated from Vcmax (µmol 24CO2 m-2 s-1, derived from Eq 1) as follows: 10 1times the nitrogen content of Rubisco (Gleadow et al., 1998) This cost (3-4 times 2Rubisco N) allows one to scale the nitrogen trade-off between photosynthesis versus 3leaf structure The shaded zone in Figure represents the upper bound of this trade4off For points falling within the shaded zone, all of leaf nitrogen would be accounted 5for by photosynthesis and cell wall material, leaving none for other cellular functions 6Our data fall below this zone, suggesting that little soluble protein inadvertently stuck 7to the cell wall pellet, leaving to 10% of leaf nitrogen unaccounted for Increased allocation of nitrogen to structure was accompanied by a reduced 9investment in Rubisco for both Polygonum (Onoda et al., 2004) and Quercus 10(Takashima et al., 2004) leaves However, for Polygonum, the slope of the 11relationship (-1.5) greatly exceeded a direct trade-off (-0.25 to -0.33) Compared to 12deciduous Quercus, evergreen Quercus leaves increased nitrogen allocated to cell 13walls and decreased nitrogen allocated to Rubisco (Takashima et al., 2004), but the 14slope (-0.56) again exceeded a direct trade-off A third study sampled leaves of 15Lindera umbellata throughout a growing season (Yasumura et al., 2006) This 16revealed that allocation of nitrogen to cell walls increased early in the season without 17any concomitant change to Rubisco and then during autumn, nitrogen released from 18Rubisco degradation was also not associated with any change in cell wall nitrogen 19Clearly there was no internal trade-off in nitrogen allocation between cell wall and 20Rubisco through the lifespan of these leaves The spread of the Eucalyptus data 21revealed no correlation between Rubisco and cell wall nitrogen (Figure 7) and the 22Acacia species pair from the ANBG actually had greater nitrogen allocation to 23Rubisco as the fraction of nitrogen allocated to cell walls increased Considering all of 24the data together reveals that the 23 species populate a large part of the space below 25the shaded zone The striking trade-off between nitrogen allocated to Rubisco versus 23 1cell walls observed for deciduous and evergreen Quercus is therefore unlikely to hold 2as a general rule Although we were only able to assay cell wall nitrogen and had to 3calculate Rubisco nitrogen from gas exchange, our results refute the generality of the 4claim that increasing amounts of cell wall nitrogen in leaves with greater LMA 5necessarily result in a reduction in nitrogen allocation to Rubisco 7ACKNOWLEDGEMENTS 8We would like to the ANBG for allowing us to sample from the gardens and 9Stephanie McCaffery for her painstaking analyses 24 1Table Leaf attributes of the congeners studied in the ANBG Values are mean ± standard error for at least four oven-dried leaves 2Abbreviations: Nm (leaf nitrogen concentration), Aa (CO2 assimilation rate per unit area), PNUE (Aa normalised to Ci of 300 µmol mol-1 divided 3by leaf nitrogen content per unit area), Ci/Ca (ratio of intercellular to atmospheric CO2) and ∆ (carbon isotope discrimination) Asterisks indicate 4statistical significance for t-tests (two tailed and assuming unequal variance) for a given attribute within each genus, where **P