Use of stable isotopes to quantify nitrogen, potassium and magnesium dynamics in young Scots pine (Pinus sylvestris)

Transcription

1 RESEARCH New Phytol. (2000), 146, Use of stable isotopes to quantify nitrogen, potassium and magnesium dynamics in young Scots pine (Pinus sylvestris) M. F. PROE*, A. J. MIDWOOD AND J. CRAIG Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB15 8QH, UK Received 11 November 1999; accepted 24 February 2000 SUMMARY Two-yr-old Scots pine (Pinus sylvestris) seedlings were grown in sand culture for 1 yr with a generous of a balanced nutrient solution. Trees were repotted into clean sand in February 1998 and given either a reduced or adequate nutrient containing enriched N, K and Mg to label nutrient uptake during spring Trees doubled their biomass during the experiment. Whole-tree net photosynthesis was reduced by 43% after 95 d in trees that received the lower nutrient (P 0 001), although differences in biomass between the two treatments were less pronounced. Remobilization contributed 83, 82 and 52% of the N, K and Mg, respectively, used to support growth of new tissues in trees that received reduced nutrient. Those receiving the higher nutrient still obtained 44 59% of nutrients used for spring growth of new tissues from remobilization. Current nutrient had no significant effect on the amount of N or Mg remobilized to new tissues but K remobilization was less in trees that received the lower nutrient (P 0 025). The importance of remobilization in young trees and problems associated with quantifying internal cycling of nutrients are discussed. Key words: Scots pine, internal cycling, N, K, Mg, stable isotopes, nutrient and uptake, nutrient remobilization. INTRODUCTION Quantifying the nutrient dynamics of trees is crucial to the understanding and prediction of the impact of environmental stress and management practices. Issues such as climate change, acid deposition and whole-tree harvesting have led to the development of more sophisticated diagnostic techniques for assessing the nutrient status of trees (Rabe, 1990; Linder, 1995; Proe et al., 1999). Many symptoms of nutritional stress have been associated with nutrient imbalance rather than straightforward deficiency of a single nutrient (Ende & Evers, 1997; Sun & Payn, 1999). The mechanisms underlying the development of nutrient imbalance can be difficult to ascertain because the factors that affect the uptake and recycling of different nutrients within whole plants are poorly understood (Marschner et al., 1997). Trees rely heavily upon the internal cycling of mobile nutrients to sustain their growth. Millard (1996) reported that 18 93% of the nitrogen *Author for correspondence (tel ; fax ; m.proe mluri.sari.ac.uk). required for leaf growth in young trees might come from remobilization. Miller (1986) calculated that 9 58% of the N, K and Mg required for growth of Corsican pine (Pinus nigra var. maritima) was derived from retranslocation and that this is unlikely to become a major source of nutrient until the tree canopy is fully formed. By contrast, Fife & Nambiar (1982) considered that internal nutrient cycling provided a mechanism to sustain rapid growth when the of nutrients from the soil was insufficient. They concluded that retranslocation of nutrients might be as important and proportionately large in young trees as it is in old ones. Studies of remobilization in trees have often suffered from flawed or intractable methodologies (Millard, 1996). Deriving whole-tree nutrient budgets is time-consuming, and such budgets are often difficult to close because of problems associated with the recovery of root systems. Seasonal sampling requires careful matching of plant components from one period to the next and often presupposes that retranslocation occurs only during leaf senescence. Direct uptake of nutrients into storage in autumn has been clearly demonstrated (Millard & Proe, 1991) as has remobilization from current needles during a

2 462 RESEARCH M. F. Proe et al. single growing season (Nambiar & Fife, 1987; Proe & Millard, 1994). The use of isotopes has enabled direct measurement of nutrient uptake and remobilization. Results have clearly demonstrated that trees can partition nutrients derived from current uptake differently from those remobilized from storage, this being shown for both P (Proe & Millard, 1995) and N (Proe & Millard, 1994). Other authors have also reported that the use of net fluxes in trees underestimates the importance of retranslocation (Mead & Preston, 1994). We have used N, K and Mg in young Scots pine (Pinus sylvestris) trees to quantify directly the contribution of uptake and remobilization to sustaining spring growth and to determine the effect of current nutrient on the amounts of N, K and Mg remobilized. The data presented allow direct comparisons to be made between net and actual flux estimations within the same trees. MATERIALS AND METHODS Experimental design Twenty 2-yr-old Scots pine (Pinus sylvestris L.) seedlings were planted 7 April 1997 in 15-l plastic pots containing fine sand with a layer of coarse grit in the base to assist drainage. Each week, 1000 cm of nutrient solution containing 6 0, 1 33, 1 25, 0 75, 2 0 and 1 25 moles m of N, P, K, Mg, Ca and S, respectively, with 20, 10, 5, 1 and 1 mmoles m of Fe, Mn, B, Zn and Cu, respectively, was applied to each tree (Millard & Proe, 1991). Additional deionized water was applied as required during summer. Trees were repotted in clean sand 11 February 1998 and deionized water was applied as necessary until 26 February 1998, when 10 trees were selected at random and harvested for determination of Table 1. Composition of labelled nutrients added to each tree 4 March 8 June 1998 Nutrient compound (mg) Nutrient treatment LOW HIGH NH NO NH NO (5 atom%) mg N per tree K SO KCl (98.7 atom%) mg K per tree MgSO.7H O MgO* (99.6 atom%) mg Mg per tree *Dissolved in HCl before addition to nutrient solution. biomass and nutrient content. On 4 March 1998 the ten remaining trees were randomly assigned to either LOW or HIGH nutrient treatments. Trees in the HIGH treatment received 600 mg N per tree, slightly less than their initial N content, and the nutrient of those in the LOW treatment was reduced to 200 mg N per tree. Other nutrients were applied to each treatment based upon the ratios to N used during Each tree received a total of 10 l of either LOW or HIGH nutrient solution, containing enriched isotopes of N, K and Mg (Table 1). Additional deionized water was added as required. Any excess solution draining from the pots was collected in plastic trays and returned to the pots. All trees were grown in a ventilated glasshouse under ambient temperature and light conditions at the Macaulay Land Use Research Institute in Aberdeen, Scotland (lat N, long W). Whole-tree net photosynthesis Measurements of whole-tree net photosynthesis were made in the glasshouse during June 1998, immediately before the trees were harvested, using an LCA-4 IRGA (Analytical Development Company Ltd, Herts, UK) connected in a closed system to a 517-l perspex cuvette (volume net of pot volume). Changes in CO concentration over 6 min were used (after correction for blanks) to calculate rates of whole-tree net photosynthesis under ambient light and temperature. Recorded PAR was used as a covariate in the data analysis. Five measurements per tree were taken and the mean rate of net photosynthesis per tree calculated for use in ANOVA. Tree harvesting and chemical analyses Ten trees were harvested 26 February 1998, when they were still dormant and before the addition of isotope. Each tree was removed from its pot and separated into buds, needles, wood, bark, coarse ( 2 mm diameter) and fine ( 2 mm diameter) roots. Sand was sieved to recover any remaining roots and all roots were washed to remove sand. A second harvest of 10 trees (five from the LOW nutrient and five from the HIGH nutrient treatments) was taken 8 June 1998, 95 d after isotope application commenced. These trees were divided into new needles, new wood, new bark, old needles, old wood, old bark, coarse roots, fine roots and new white roots. All new tissues were known to have been produced during the current growing season. Once again, the sand was sieved to remove any remaining roots and all roots were washed. Samples were freeze-dried, weighed and milled before analysis. Total N and N enrichment were determined using a TracerMAT continuous flow,

3 RESEARCH Nutrient dynamics in Scots pine 463 Table 2. Dry weight and nutrient content at the start of the experiment in February 1998 ( SE, n 10) and effect of nutrient on mean tree dry weight and initial nutrient content, estimated from unlabelled nutrient content in trees harvested in June 1998 Time of harvest Nutrient treatment D. wt (g per tree) N content (mg per tree) K content (mg per tree) Mg content* (mg per tree) Feb 1998 None June 1998 Low (43) High (43) SED (4) *Values in parentheses are after correction (see the Materials and Methods section) for uptake of unlabelled Mg from the sand. Standard error of difference (SED) between treatment means in June 1998 based upon 5 replicates. isotope ratio mass spectrometer (FinniganMAT, Hemel Hempstead, UK). Total K and Mg were determined by ICP-AES of acid-digested samples (Midwood et al., 2000). Ratios of K: K and Mg: Mg were determined using a VG354 thermal ionization mass spectrometer (Micromass Ltd, Wythenshawe, UK) (Midwood et al., 2000). Predicted Mg content per tree (mg) Nutrient uptake and remobilization The amount of excess (i.e. above background) N, K and Mg in each tree harvested in June was used to estimate nutrient uptake from the solutions applied to that tree: uptake (excess t excess s ) total s (uptake, nutrient content in tissue derived from applied solution (mg per tree); excess t, excess isotope in tissue (µg); excess s, excess isotope in total solution applied to each tree (µg); total s, total nutrient applied to each tree (mg per tree)). When trees were harvested in June, the difference between their total nutrient content and the amount of nutrient taken up by each tree provided an estimate of the initial nutrient content of each tree at the start of the year. This represents the maximum size of pool of nutrients that can contribute to remobilization. At a whole-tree level, the estimated mean initial contents of N and K derived from the isotope data agreed closely with the mean values obtained for the 10 trees harvested before application of isotope but this was not the case for Mg (Table 2). The use of isotope data substantially overestimated the initial Mg content, when compared with the mean value for trees harvested in March, suggesting that additional Mg was taken up by the trees that was not isotopically labelled. It is likely that this was present in the sand used to re-pot the trees. To overcome this problem and provide an estimate for the initial pool of Mg in each tree (rather than apply the mean value recorded in March to every tree), regression analysis was used. A regression of Measured Mg content per tree (mg) Fig. 1. Predicted Mg content of 10 trees harvested in February 1998 based on multiple regression from N and K contents compared with measured Mg content. total Mg content on total N and K contents for the 10 trees harvested February 1998 was used to predict the initial Mg content of the 10 trees harvested June 1998 from their initial N and K contents derived from the N and K isotope analyses. This regression explained 74% of the variation of initial Mg content and two trees were observed to be outliers to the regression model (Fig. 1). If these were excluded from the regression then 99% of variation was explained by the model, based upon the eight remaining trees and the form of the regression was as follows: Mg i ( N i ) ( K i ) 9 5 (Mg i, initial Mg content before isotope application (mg Mg per tree); N i, initial N content before isotope application (mg N per tree); K i, initial K content before isotope application (mg K per tree)). The mean estimate for initial Mg content derived from this method agreed closely with the mean value obtained directly from the 10 trees sampled in February (Table 2) but allowed individual estimates to be made for each tree. of Mg per tree was calculated as the difference between the total content at harvest June 1998 and the initial content estimated for February was assumed to be distributed between tissues in the same proportion

4 464 RESEARCH M. F. Proe et al. as the excess Mg label, and the unlabelled content of each tissue calculated as the difference between total content in that tissue and the uptake to the same tissue. For new growth, remobilization of N, K and Mg was calculated as the difference between the total nutrient content and nutrient uptake within each tissue. In older tissues, nutrient that was not derived from uptake represented the initial pool of nutrient present in the tree before application of isotope. Statistical analysis For each tissue, or group of tissues, ANOVA was used to estimate treatment effects for the amount of nutrient applied (LOW vs HIGH) and to compare differences between the amounts of nutrients obtained from each source (uptake vs remobilization) for each of the three nutrients (N, K and Mg). Where necessary, Log e transformations were used for analysis of ratios between nutrients obtained from remobilization and from uptake within specified tissues. For gas exchange measurements, ANCOVA was used with PAR as the covariate. RESULTS Tree growth and nutrient uptake During the experiment the mean total dry weight per tree doubled, with no significant difference between treatments (Table 2). Approximately two thirds of biomass increment occurred as new tissues (needles, wood, bark and white roots), with one third being added to existing tissues. By 8 June 1998, mean tree height and root collar diameter were 57 cm and 1 4 cm, respectively, and were not affected by nutrient during the 95 d that treatments were applied. Whole-tree net photosynthesis measured in June was reduced by 43% in trees that received the lower nutrient (Table 3). No significant treatment effect was observed when the rate of net photosynthesis was expressed per unit needle weight, per unit needle N or Mg content. However, whole-tree net photosynthesis per unit needle K content increased by 46% (P 0 016) in trees receiving the lower nutrient (Table 3). At the end of the experiment new needle biomass was reduced by 25% (P 0 005) in trees receiving the lower nutrient (Table 4) and accounted for 54 and 60% of total needle biomass in the LOW and HIGH treatments, respectively. Total N and K contents were reduced by 19 and 33%, respectively, in the LOW treatment but nutrient had no significant effect on total Mg content per tree (Table 4). Nutrient concentrations expressed on a wholetree or new-needle basis were lower in the trees receiving the lower nutrient for N and K but did not differ significantly for Mg (Table 4). Within the trees harvested before isotope application, 5% of total nutrient content was recovered from buds. Nitrogen uptake accounted for 13 and 31% of total N content in LOW and HIGH nutrient treatments, respectively (Table 5; P 0 001). Equivalent values for K uptake were 23 and 45% (P 0 001), but there was no significant treatment effect on the proportion of Mg content per tree that was derived from current uptake (49%; Table 5). Nutrient allocation to new components The use of stable isotopes allowed a distinction to be made between the sources of nutrients used to support the growth of new components (new needles, new wood, new bark and white roots). Remobilization provided a greater proportion of nutrients used to support growth of new components in trees that received the lower nutrient (P 0 001; Fig. 2). This difference was less apparent (though still significant) for Mg than for N and K (P 0 041). Remobilization contributed a smaller proportion of Mg used to support growth of new tissues than of N and K (Fig. 2). This was most apparent at the lower nutrient (P 0 001) although, apart Table 3. Effect of nutrient on net photosynthesis in June 1998 expressed per tree, per unit needle biomass (g per tree) or per unit needle nutrient content (mg per tree) Net photosynthesis (µg CO s ) Nutrient treatment LOW HIGH SED P Tree Needle biomass (g) Needle N content (mg) Needle Mg content (mg) Needle K content (mg) Standard error of difference between treatment means (SED) and probability value (P) from ANOVA based upon 5 replicates and with PAR as covariate.

5 RESEARCH Nutrient dynamics in Scots pine 465 Table 4. Effect of nutrient on dry weight and nutrient concentration of new needles and nutrient content per whole tree Nutrient treatment LOW HIGH SED P Current needles Dry weight (g) N (%) K (%) Mg (%) Total tree N content (mg) K content (mg) Mg content (mg) Standard error of difference between treatment means (SED) and probability value (P) from ANOVA based upon 5 replicates. Table 5. Effect of nutrient on N, K and Mg uptake (mg per tree) and uptake as a proportion of total nutrient content (mg) Nutrient treatment LOW HIGH SED P Nitrogen Per tree % of total Potassium Per tree % of total Magnesium Per tree ns % of total ns Standard error of difference between treatment means (SED) and probability value (P) from ANOVA based upon 5 replicates. Remobilization:uptake Remobilization:uptake (a) Needles Wood Bark Roots (b) Needles Wood Bark Roots from new roots, the difference was also significant for trees that received the higher nutrient (P 0 001). Remobilization contributed a smaller proportion of N and K contained within new roots than in new shoot components, irrespective of the level of nutrient (Fig. 2; P 0 001). This was not the case for Mg at either of the two rates. Contribution of remobilization to support of growth of new tissues Nutrient had no significant effect on the amount of N remobilized, which accounted for 39 and 43% of the original N content in LOW and HIGH treatments, respectively (Fig. 3). By 8 June, remobilized N had contributed 83 and 59% of the total N content in new tissues of trees from LOW and HIGH treatments, respectively (P 0 001). Remobilization:uptake (c) Needles Wood Bark Roots Fig. 2. Effect of nutrient upon the ratio of nutrient derived from remobilization to that derived from root uptake in new growth. (a) N. (b) K. (c) Mg. Filled columns, trees receiving the higher nutrient ; open columns, those receiving the lower nutrient. Error bars, 1 SE with 5 replicates per treatment. During the experiment, N uptake to old tissues had replaced 18 and 35% of N that had been remobilized to new tissues in LOW and HIGH treatments, respectively (P 0 001).

6 466 RESEARCH M. F. Proe et al. Low nutrient 57 (7) mg N per tree to new tissues 218 (15) High nutrient 343 (34) New tissues 528 (20) 286 (28) Retranslocation from old to new tissues 310 (8) 501 (34) Old tissues 520 (52) 52 (2) to old tissues Fig. 3. Effect of nutrient on N uptake (straight arrows), remobilization (curved arrows) and pool sizes (boxes) in old (shaded) and new (unshaded) tissues (mg N). Values in parentheses represent 1 SE with 5 replicates per treatment. 108 (19) Low nutrient 30 (3) mg K per tree to new tissues 116 (3) High nutrient 164 (12) New tissues 280 (5) 134 (9) Retranslocation from old to new tissues 164 (6) 194 (12) Old tissues 252 (20) 52 (3) to old tissues Fig. 4. Effect of nutrient on K uptake (straight arrows), remobilization (curved arrows) and pool sizes (boxes) in old (shaded) and new (unshaded) tissues (mg N). Values in parentheses represent 1 SE with 5 replicates per treatment. 126 (12) Increasing nutrient increased the amount of K remobilized to support growth of new tissues (P 0 025) from 49% of the original K content in trees receiving the lower nutrient to 57% in the HIGH nutrient treatment (Fig. 4). Remobilized K had contributed 82 and 59% of the K content of new tissues by 8 June (P 0 001), similar to the contribution for N. During the experiment K uptake to old tissues replaced 39 and 77% of K remobilized to new tissues (P 0 001). Current nutrient had no significant effect on the amount of Mg remobilized, which accounted

7 RESEARCH Nutrient dynamics in Scots pine 467 Low nutrient 16 (1) mg Mg per tree to new tissues 23 (1) High nutrient 33 (2) New tissues 41 (1) 17 (2) Retranslocation from old to new tissues 18 (1) 50 (4) Old tissues 49 (7) 23 (2) to old tissues Fig. 5. Effect of nutrient on Mg uptake (straight arrows), remobilization (curved arrows) and pool sizes (boxes) in old (shaded) and new (unshaded) tissues (mg Mg). Values in parentheses represent 1 SE with 5 replicates per treatment. 24 (5) for 39 and 43% of the original Mg content in LOW and HIGH treatments, respectively (Fig. 5). Remobilized Mg had contributed 52 and 44% of the Mg content of new tissues by 8 June (P 0 180). During the experiment, Mg uptake to old tissues was greater than the amount remobilized in both treatments. DISCUSSION Total tree biomass doubled during the present experiment but the effects of nutrient were masked by the initial tree size at the start. Growth of new tissues was reduced in the LOW nutrient treatment and this was confirmed by the large treatment effect on whole-tree net photosynthesis that developed as the experiment progressed. Similar results have been reported for the spring growth of Sitka spruce subjected to contrasting supplies of N (Proe & Millard, 1994) or P (Proe & Millard, 1995), where current nutrient had little effect on total tree biomass for several months. of Mg, in addition to that added in the nutrient solution was probably from sources in the sand at the start of the experiment, as has been reported by Hogberg et al. (1995) when growing Scots pine seedlings in acid-washed sand. Provided that the distribution of Mg uptake reflected that for total Mg uptake, our estimates for uptake and remobilization of Mg to different tissues should remain valid. Treatment effects on nutrient concentrations in new needles suggest that N and K levels were reduced in the LOW treatment but that Mg levels were unaffected. N is closely coupled to plant growth as it is a major constituent of proteins and amino acids, in addition to being central to the processes of photosynthesis (Katzensteiner & Glatzel, 1997). Potassium is less directly involved in plant growth, having major roles in osmoregulation, enzyme activation and carbohydrate translocation (Ericsson &Ka hr, 1993; Jokela et al., 1997). The observed increase in photosynthesis per unit needle K in trees that received the lower nutrient reflects the looser coupling between growth and K in Scots pine. Many authors have used budgets from sequential samplings to estimate the contribution of remobilization to support new growth in pine trees. Miller (1986) reported that 17% of N, 24% of K and 9% of Mg required for growth of 2-m-high Corsican pine was derived from retranslocation. Helmisaari (1995) obtained similar estimates of 30, 17 and 4% for N, K and Mg, respectively, in 2-m-high Scots pine. Both authors reported that the contribution from remobilization increased substantially as stands aged, and Miller concluded that remobilization was likely to be of minor importance in young trees in which litterfall was negligible and foliage biomass was increasing exponentially. Our results call into question such interpretation, because the use of multiple stable isotopes allows a distinction to be made between nutrient uptake and remobilization and, therefore, a move away from the use of net fluxes. During spring growth of our Scots pine trees,

8 468 RESEARCH M. F. Proe et al. as their biomass doubled, remobilization contributed 83% of the N, 82% of the K and 52 of the Mg required to support this growth in trees receiving the lower nutrient. Even in the trees that received the higher nutrient, remobilization still contributed 59, 59 and 44% of the N, K and Mg, respectively, used to support spring growth of new tissues. The use of net fluxes would have underestimated the contribution of remobilization substantially. For example, for trees that received the lower nutrient (Fig. 3) we have estimated that retranslocation provided 286 of the 343 mg N recovered in the new tissues (83%). This is greater than the retranslocation that would have been predicted based on the net change in N content of old tissues estimated to be 234 mg N, (286 [N retranslocated from old tissues] 52 [N uptake into old tissues] ) or 68% of the N contained in new tissues. Using our own isotope data, uptake replenished 18 77% of the N and K remobilized from old tissues and exceeded the amount of Mg remobilized. Mead & Preston (1994) reported that net flux provided a poor measure of retranslocation from needles of 8-yr-old lodgepole pine (Pinus contorta). Their use of N revealed a 12 5% underestimate of N retranslocation in current needles and a 24% underestimate in 1-yr-old needles. The low values for the contribution of Mg retranslocation reported elsewhere might also reflect the measurement of net fluxes. Use of isotopes allows the effect of nutrient upon remobilization to be quantified a serious problem where uptake and remobilization cannot be distinguished. Our results for N confirm results from studies on other species where current nutrient had no significant effect on the amount of N or P remobilized to support new growth (Millard & Proe, 1991; Proe & Millard, 1994, 1995). The same observation held for Mg in the present study, but must be interpreted with caution, because control of Mg was poor due to uptake of Mg from the sand used in the experiment. In the case of K, however, increasing the nutrient increased the amount of K remobilized from 49 57% of initial content. This might reflect the contrasting physiological roles of N and K and the greater proportion of K available for remobilization. In a series of experiments on radiata pine, Nambiar & Fife (1991) concluded that remobilization depended upon the sinks provided by new growth rather than the of N available from the soil. It is possible that K is more sensitive than N to the increased sink strength associated with increasing nutrient. The present study has focused upon the period of spring growth in young Scots pine and has indicated the importance of remobilization to sustain growth during this time. As the season progresses, remobilization is likely to subside and uptake become increasingly important for continued growth and formation of reserves for use in the following season. In young trees, however, vigorous spring growth is important for their successful establishment and survival, often under exacting environmental conditions, and when subjected to competition from other vegetation. ACKNOWLEDGEMENTS This work was supported by the Scottish Executive Rural Affairs Department. We are grateful to Jennifer Harthill and Dawn Morley for assistance with isotope analyses and to Barry Thornton for helpful comments on the manuscript. REFERENCES Ende H-P, Evers FH Visual magnesium deficiency symptoms (coniferous, deciduous trees) and threshold values (foliar, soil). In: Huttl RF, Schaff W, eds. Magnesium deficiency in forest ecosystems. Dordrecht, The Netherlands: Kluwer Academic Publishers, Ericsson T, Ka hr M Growth and nutrition of birch seedlings in relation to potassium rate. 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