Jann P. Conroy*, Paul J. Milham, Malcolm L. Reed, and Edward W. Barlow. School of Biological ..... tosynthetic area in young plants accelerates production of.
Plant Physiol. (1990) 92, 977-982
Received for publication July 25, 1989 and in revised form October 25, 1989
0032-0889/90/92/0977/06/$01 .00/0
Increases in Phosphorus Requirements for C02-Enriched Pine Species' Jann P. Conroy*, Paul J. Milham, Malcolm L. Reed, and Edward W. Barlow School of Biological Sciences, Macquarie University, N.S.W., 2109, Australia (J.P.C., M.L.R., E.W.B.); N.S. W. Agriculture and Fisheries, N.S. W., 2116, Australia (P.J.M.) ABSTRACT
whole plant level is that other factors, such as nutrient supply, may become limiting. In the majority of CO2 enrichment studies, the foliar nutrient concentrations have not been measured. Even when they were, the significance of the data is doubtful because the levels required to produce maximum growth at elevated CO2 have not been determined. However, a study with cucumber (Cucumis sativus) indicated that nutrient requirements may be increased by high CO2 (18). Nutrient availability is particularly relevant to the potential impact of atmospheric CO2 levels on tree growth because forests are typically restricted to infertile sites. P deficiency is of special interest because it eliminates the growth response to high CO2 in crop plants (9) and P. radiata (6). We report the growth of three Pinus genotypes (two halfsib families of P. radiata and an open-pollinated family of P. caribaea) at seven levels of P and at CO2 concentrations of either 340 or 660 uL CO2 L-'. Both the P. radiata families are known to produce more dry weight at elevated CO2 (7, 8). Photosynthesis, sugar, and starch content of the needles and the level of mycorrhizal infection of the roots are also reported. The results clearly demonstrate increased P requirement of pines at elevated CO2.
Pinus radiata D. Don (half-sib families 20010 and 20062) and Pinus caribaea var hondurensis (an open-pollinated family) were grown for 49 weeks at seven levels of phosphorus and at CO2 concentrations of either 340 or 660 microliters per liter, to establish if the phosphorus requirements differed between the CO2 concentrations and if mycorrhizal associations were affected. When soil phosphorus availability was low, phosphorus uptake was increased by elevated CO2. This may have been related to changes in mycorrhizal competition. When the phosphorus concentration in the youngest fully expanded needles was above 600 milligrams per kilogram the shoot weight of all pine families was greater at high CO2 due to increases in rates of photosynthesis. More dry weight was partitioned to the stems of P. radiata family 20010 and P. caribaea. At foliar phosphorus concentrations above 1000 milligrams per kilogram (P. radiata) and 700 milligrams per kilogram (P. caribaea), growth did not increase at 340 microliters of CO2 per liter. Soluble sugar levels in the same needles mirrored the growth response, but the starch concentration declined with increasing phosphorus. At 660 microliters of CO2 per liter, shoot weight and soluble sugar concentrations were still increasing up to a foliar P concentration of 1800 milligrams per kilogram for P. radiata and 1600 milligrams per kilogram for P. caribaea. The starch concentrations did not decline. These results indicate that higher foliar phosphorus concentrations are required to realize the maximum growth potential of pines at elevated CO2.
MATERIALS AND METHODS Plant Culture
Soil was collected from a Pinus radiata plantation. It had a low level of available P and a high level of mycorrhizal inoculum. Other properties of this soil were previously described (8). There were ten pots for each of the seven P treatments planted with each Pinus family. Every pot contained 950 g of dry soil. P (CaHPO4) was added to the soil at the following rates (mg P kg-' soil): 0 (P0), 46 (P,), 92 (P2), 182 (P3), 274 (P4), 456 (P5), and 1368 (P6). Fertilizers other than P were added as previously described (6) and the soil and the fertilizers were mixed. Soluble P was added at 5 mg kg-' as NH4H2PO4 to all treatments at w2. Thereafter it was added on a regular basis to the P4, P5, and P6 treatments. N was applied as KNO3, Ca(NO3)2 .4 H20, Mg(NO3)2 .6 H20, (NH4)2SO4, and NH4NO3. The timing of these additions and the quantities of the various salts to be used were estimated from the fertilizer history and the chemical composition of the needles. Two half-sib families of P. radiata D. Don, 20010 (F10) and 20062 (F62) (Forestry Commission of N.S.W., Australia) and an open-pollinated family of P. caribaea var hondurensis
If trees grow faster at high C02, they could partially counteract the rising atmospheric CO2 levels by increasing the flux of CO2 from the atmosphere. Conifers could prove particularly important because they constitute a major proportion of the extensive boreal forests, whose area is predicted to increase with the climatic changes expected to accompany the rise in atmospheric CO2 concentration (17). Pinus radiata and Pinus caribaea were chosen as experimental material because they are the major plantation conifers in Australasia. The rate of needle photosynthesis and therefore the growth of C3 plants is limited by CO2 availability at the current atmospheric concentration of 340 qL L-'. Consequently, increasing the atmospheric CO2 concentration to 660 ,uL L-' approximately doubles both the CO2 concentration at the site of fixation and the rate of photosynthesis (22). However, dry matter production is only increased by about 40% (1 1). One possible explanation for the smaller response at the 'Supported by the Rural Credits Development Fund of Australia. 977
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CONROY ET AL.
(Batch T43 1, Department of Forestry, Queensland, Australia) were grown from seed with two uniform seedlings in each pot. Half of the pots at each P level, for each Pinus family, were placed in one growth chamber at 340 ,uL CO2 L-' and the other half in another chamber at 660 ,uL CO2 L-', i.e. there were five pots (replicates) for each treatment. Growth conditions were day/night, 25°C/18°C, 16/8 h and a PPFD measured at the top of the plants of 450 ,umol m-2 s-1. In the first experiment, P. radiata FIO and P. caribaea were grown at seven levels of P (Po-P6). In the second experiment P. radiata F62 was grown at seven levels of P (PO-P6). FO0 was also grown at three P levels (P0, P3, and P6) to provide a link between the experiments. More soluble P was added to the P4 to P6 treatments in the second experiment. Dry weight
At w24 and w49 in both experiments, the shoots of one plant per pot were harvested and separated into foliage and stems. These components were dried and weighed. Mycorrhiza At w49 in the first experiment, the roots of P. radiata FlO from the ten replicates of the Po, P2, and P6 treatments were washed free of soil over a 0.8 mm sieve and examined for
mycorrhiza. The number of mycorrhizal root tips per root system was counted on the fresh roots. Mycorrhizal status was confirmed by staining the roots with Ponceau Red and examining them under a light microscope. Where mycorrhizas with different structures were found the root tips were sectioned and the characteristics of the mycorrhizas were noted. Chemical Analysis
Sampling was carried out immediately prior to the commencement of a light period. At w24, foliage (juvenile leaves plus needles) was sampled and composited within treatments and at w49 the youngest fully expanded needles were sampled from each pot. The samples were frozen in liquid N2, freeze dried, and ground. Subsamples were chemically analyzed for N, P, K, Ca, Mg, Mn, Cu, Zn, Fe, Cl, S, and B as described previously (8). For carbohydrate analysis, duplicate subsamples were extracted twice in ethanol/water (4/1, v/v) at 80°C and once with boiling water. The ethanolic extracts were combined and evaporated to dryness, then the sugars were dissolved in water. The starch in the extracted solid residues was hydrolyzed by boiling in 0.2 M KOH. Sugars in the three extracts were measured as glucose after reaction with anthrone (19). The ethanol and water soluble fractions were summed to give total soluble sugars.
Photosynthesis Room temperature Chl a fluorescence of needles of each genotype was measured at w24 and w49 in both experiments. Two of the youngest fully expanded fascicles were removed from each plant. The tips and bases of the needles were
Plant Physiol. Vol. 92,1990
discarded and the remaining portions cut into 15 mm lengths. A sample from each pot was prepared by laying the 15 mm lengths parallel and adjoining one another on a plastic disc. The needles were held in place with double-sided adhesive tape, with their adaxial surfaces exposed. Samples were wrapped in plastic film to prevent moisture loss and were dark adapted for at least 1 h. Chl a fluorescence was measured as previously described (5). At w49, A2 was measured on at least two groups of three of the youngest fully expanded needles from each pot using a portable LCA2 IR gas analyzer system in the open configuration (Analytical Development Co. Ltd., Hoddesdon, UK). The temperature and VPD in the chamber were not controlled and were 25 to 27°C and 15 to 20 Pa KPa-', respectively. The CO2 concentration in the air entering the leaf chamber (6 mL min-') was either 340 or 660 ,uL L-'. Illumination was provided by a 200 W daylight floodlight (RDS model UFH-2, HMI 200, Ryudensha Co. Ltd., Japan) to give a PPFD of 800 to 900 Imol m-2 s-'. Data Analysis Treatment effects were assessed by analysis of variance. LSDs were calculated from the error mean squares. The P response curves were fitted by inspection. RESULTS Nutrition
The youngest fully expanded needle is the foliar component we used to assess the nutritional status of Pinus species. The P concentration in these needles at w49 was 600 to 1900 mg kg-' for Pinus radiata and 450 to 1440 mg kg-' for Pinus caribaea at the seven P levels (PO-P6). The CO2 treatments had no effect on P concentration. The foliar P concentrations of P. radiata F1O grown at the P4 to P6 treatment levels were higher in experiment 2 because the amount of soluble P added was increased in this experiment. For all treatments, nutrients
other than P fell within the foliar concentration ranges considered to be adequate to sustain the genetic potential for growth at 340 ,uL CO2 L-1 (2, 24). Growth and Partitioning of Dry Weight
Both higher P and CO2 levels increased the weight of the shoots (foliage and stems) of all Pinus families and the effects were synergistic (Fig. 1, a, b, c). For the same treatments in the two different experiments, the growth of shoots of P. radiata F1O was not significantly different (P 6 0.05), e.g. dry weights at P6 were 21 and 41 g for the 340 and 660 uL C02 L-' treatments, in the first experiment, and 20 and 43 g in the second experiment (Fig. lb). This similarity enabled valid comparisons of the response of the Pinus genotypes to the treatments to be made between experiments. The response to increasing P availability differed at the two Abbreviations: A, net CO2 assimilation rates; RUBPcase, ribulose 1,5-bisphosphate carboxylase; F0, constant yield Chl fluorescence; Fv, variable yield Chl fluorescence; Fp, maximum Chl fluorescence (constant plus variable yield fluorescence). 2
PHOSPHORUS REQUIREMENT AT ELEVATED CO2
979
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