Trees (2005) 19: 213–223 DOI 10.1007/s00468-004-0384-1
ORIGINA L ARTI CLE
Marjoriitta Möttönen . Tarja Lehto . Hannu Rita . Pedro J. Aphalo
Recovery of Norway spruce (Picea abies) seedlings from repeated drought as affected by boron nutrition Received: 2 June 2004 / Accepted: 8 September 2004 / Published online: 16 November 2004 # Springer-Verlag 2004
Abstract The effects of two boron (B) levels on growth, shoot water potential, gas exchange and nutrient accumulation in Norway spruce [Picea abies (L.) Karst.] seedlings were studied in a growth room experiment lasting 22 weeks which included well-watered control seedlings and seedlings exposed to one (8 days) or two (6+8 days) periods of drought and a rewatering period (8 days) at the end of the experiment. The effects of B and drought were monitored during drought and recovery. Needle B concentrations were 6 mg kg−1 (−B treatment) and 34 mg kg−1 (+B treatment) at the end of the experiment. The −B seedlings showed visible symptoms of damage in the upper shoot after repeated drought and had reduced height growth, root dry mass, allocation of biomass to roots and formation of root tips and mycorrhizas and reduced needle P, Ca, and Mg concentrations and contents. In contrast, 15N uptake, shoot water potential and gas exchange were not markedly affected by B. It can be concluded that the visible symptoms of damage at low B were probably related to reduced B transport due to repeated drought. In contrast, the effects of low B on growth, particularly of the roots, and on nutrient uptake can be regarded as early effects which occur before any influence on shoot water potential or gas exchange. The
M. Möttönen (*) . T. Lehto . P. J. Aphalo Faculty of Forestry, University of Joensuu, P.O. Box 111, 80101 Joensuu, Finland e-mail:
[email protected] Tel.: +358-13-2514423 Fax: +358-13-2513590 H. Rita Department of Forest Resource Management, University of Helsinki, P.O. Box 27, 00014 Helsinki, Finland Present address: P. J. Aphalo Department of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, 40351 Jyväskylä, Finland
positive effects of B on root biomass and nutrient accumulation are of particular importance regarding the establishment of young seedlings in the field. Keywords Boron . Drought . Gas exchange . Growth . Recovery
Introduction Boron (B) deficiency is the most widespread micronutrient problem in forest trees worldwide (Stone 1990). In Finland and Scandinavia B deficiency has been widely reported in Norway spruce [Picea abies (L.) Karst.] stands (Brække 1977, 1979; Aronsson 1983; Silfverberg 1980; Hynönen et al. 1999). The importance of B, especially for root growth, has recently been documented in Norway spruce seedlings (Möttönen et al. 2001a,b) and in mature Norway spruce trees in the field (Lehto 1994; Möttönen et al. 2003). Roots play a major role in the regulation of water and nutrient uptake, and in the maintenance of plant water balance. Therefore, an extensive and efficient root system is important in drought resistance of plants. Little research has been focused on the interactive effects of low B and drought stress in tree seedlings, however. We found earlier that the height growth of Norway spruce seedlings at low B was reduced when they were exposed to a drought period of 9 days, whereas at adequate B it was not affected by drought (Möttönen et al. 2001a). The seedlings grown at adequate B had lost water more rapidly than those at low B, when measured on the 4th day of drought, however, and had earlier stomatal closure and decreased photosynthesis (Möttönen et al. 2001a), but it remained unknown whether low B depresses water uptake during the first days of drought, before the 4th day. In addition, the effect of B after repeated periods of drought was not studied. Under field conditions in particular, seedlings would benefit if they could resist repeated droughts, but it is possible that seedlings grown at low B may recover from drought more slowly than those grown at adequate B. In fact, the effect of B nutrition on recovery
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from drought has not been studied before, although the ability to recover quickly would be important for seedling survival. In addition, while several studies have examined severe B deficiency in plants, little emphasis has been placed on the effects of only moderately low B levels. We therefore set out here to examine Norway spruce seedlings having low and adequate needle B concentrations. We hypothesized that the Norway spruce seedlings with adequate internal B would be able to resist drought better on the first few days than those with low internal B, that the seedlings with low internal B exposed to two periods of drought would suffer most, and that those with low internal B would recover from drought more slowly. To test these hypotheses, we measured growth, shoot water potential, gas exchange, and nutrient accumulation in seedlings with low and adequate internal B during drought and recovery periods. Nitrogen uptake after drought was determined by feeding the seedlings with 15N.
Materials and methods Experimental design and cultivation of the seedlings The experiment followed a complete randomized block design with two factors, boron (B) and drought (D). The boron factor had two levels, −B: no B supplied, and +B: adequate B supply rate, and drought three levels, D0: no drought, D1: one period of drought, and D2: two periods of drought. There were six blocks (true replicates), each having 35 seedlings in individual pots in each of the six (2×3) treatments, resulting in 1,260 seedlings altogether. The blocks were surrounded by border seedlings that were not used for the measurements. The seedlings were allocated to blocks and treatments within the blocks at random. Norway spruce seeds of a southern Finnish provenance from a seed orchard were soaked in cold water overnight, treated with 30% H2O2 for 15 min and then rinsed five times with distilled water before sowing in 263 cm3 pots (Deepots D-16, Stuewe, Corvallis, OR, USA), four seeds per pot, in a substrate containing 70% (vol) quartz sand of particle size 0.5–1.5 mm and 30% (vol) sieved mor humus from a low-B Norway spruce forest (Lehto and Mälkönen 1994). The initial total B concentration in the soil mixture was low (0.45 mg kg−1, determined by the azomethine-H method). The seedlings were grown in a walk-in growth room (Conviron GR77, Controlled Environments, Winnipeg, MB, Canada) for 156 days (22 weeks). They were thinned to one per pot 14 days after sowing, when all of the seeds had germinated. This day is referred throughout as day 1. The day length (photoperiod) in the growth room was 20 h, with a day/night temperature regime of 20/15°C, cooling/warming at 5°C h−1 and relative humidity of 70/80% (water vapor pressure deficit 7.0/3.4 mmol mol−1). Day/night irradiance was 350/0 μmol m−2 s−1 PAR from incandescent lamps (60 W, Oy Airam, Finland) and fluorescent tubes (VHO 215 W, Sylvania Cool White, Sylvania, USA). The red:far-red photon ratio was 1.16
(LI-1800 spectroradiometer, Li-Cor, Lincoln, NE, USA). Light intensity was reduced/increased in three 1-h steps. The pots were watered daily with deionized water (containing no B when measured directly by the azomethine-H method) until day 9, after which the seedlings were fertilized with a nutrient solution (Riddoch et al. 1991) to make sure that they would not suffer other nutrient imbalances in addition to the low B (−B treatment). One-half of the seedlings were fertilized with a complete solution (adequate B supply, +B) and the other half with a B-free solution (low B supply, −B). The proportion of B:N in the +B solution was 1:1,000. Except for B, the proportions of the nutrients did not vary between the treatments, the proportions of N, P, and K being 100:16:55 by weight, the ratio of nitrate-N to ammoniumN 7:5 and pH 4.0. The nutrient solution used for the first fertilization (day 9) contained 1.07 mg N dm−3 and 1.10 μg B dm−3 in the +B treatment (no B in the −B treatment), after which the amounts of nutrients were increased each time to meet the requirements for a relative growth rate (RAR) of 6% per day (except for B concentration in the −B treatment). The seedlings were fertilized three times a week with an excess of nutrient solution (15 ml per seedling on days 9–86, 20 ml per seedling on days 88–105, 25 ml per seedling on days 134– 142) to ensure that all the growth medium was flushed with fresh solution. One-third of the seedlings in both B treatments were subjected to a drought period of 6 days beginning on day 118, after which, from day 124 onwards, they were watered with 30 ml deionized water for 2 days and then subjected to a second drought period of 8 days. These seedlings will be referred to below as D2 seedlings (exposed to two drought periods). Another third of the seedlings were subjected only to the later drought period of 8 days, and they will be referred to as D1 seedlings, while the remaining third were watered daily (15 ml deionized water per seedling) and will be referred to as D0 seedlings (no drought). The D0 seedlings received only deionized water from day 118 to day 134, as did the D1 seedlings and from day 118 to day 126. After the drought treatments ended (day 134) all the seedlings were fertilized with the nutrient solution four times (25 ml) during a recovery period of 8 days. The nutrient solution used for the last fertilization (day 142) contained 410 mg N dm−3 and 0.41 mg B dm−3 in the +B treatment (no B in the −B treatment). Four seedlings were randomly selected among the 35 per treatment in each of the six blocks for the growth measurements (height and dry mass) on day 134 (after drought) and 142 (after recovery). Root tips and mycorrhizas were counted from a half of these seedlings (two seedlings per treatment and block on each day). Shoot water potential and gas exchange (net CO2 exchange rate, stomatal conductance and intercellular CO2 concentration) were measured in one seedling representing each treatment and block on days 2, 3, and 5 of the later drought period and also on days 1, 2, 5, and 8 of the recovery period.
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Growth, root tips, mycorrhizas and visible damage The height of the seedlings was measured with a ruler, the dry mass of the needles, stems and roots was determined (at 60°C with forced ventilation for a minimum of 3 days), and the allocation of the biomass to different parts of the plants (roots, stems and needles) was reported as proportions of the total dry mass. Root tips and mycorrhizas were counted and classified according to their morphology and color under a stereo microscope. The root system was cut into pieces approximately 3 cm in length which were divided equally between six petri dishes, two of which were selected at random for counting. The numbers of root tips and mycorrhizas in the whole root system were then estimated based on the results of the counting of the two subsamples and the measurements of dry mass. The root tips were separated into (a) long root tips (roots of unlimited growth, relatively long, sparsely branched, and with a sharp tip), (b) non-mycorrhizal short roots (short roots of limited growth, no mantle, no further branching), (C) mycorrhizal short roots (short roots of large diameter relative to length, with a fully developed mantle), (d) Cenococcum geophilum Fr. mycorrhizas (short roots of large diameter relative to length, with a characteristic black mantle), and (e) developing mycorrhizas (short roots of large diameter relative to length, mantle not fully developed, de la Rosa et al. 1998). The occurrence of visible symptoms of damage in the upper shoot was recorded for all the seedlings on day 134, at the end of the second drought cycle, using the following classification: (1) healthy, with no visible symptoms of damage, (2) mild visible symptoms of damage, with some yellow or brown needles, (3) medium visible symptoms of damage, with several yellow or brown needles, and (4) severe visible symptoms of damage with dead apical tips and abundant yellow or brown needles.
ments were performed inside a growth chamber (PGW 36, Conviron, Controlled Environments, Winnipeg, MB, Canada) where the relative humidity was 70%, air temperature 20°C, water vapor pressure deficit 7.0 mmol mol−1 and PAR 700 μmol m−2 s−1(according to preliminary tests light saturation of photosynthesis for the well-watered seedlings occurred at or below this PAR value). The needle temperature was 21°C and the ambient CO2 concentration (that in the sample analyzer) 350 μmol mol−1. The vapour pressure deficit at the leaf surface was 1.0–1.2 mmol mol−1. The projected needle areas obtained by scanning the needles (ScanJet 6100 C/T, Hewlett Packard, Palo Alto, CA, USA) and analyzing the images with WinNEEDLE 3.0.1 (Régent Instruments, Québec City, Canada) were used in the calculations. The seedlings with severe visible damage were not included in the measurements. Nutrients Boron concentrations were determined by dry ashing and azomethine-H determination, and N concentrations by the Kjeldahl method (Halonen et al. 1983). Concentrations of K, Ca, Mg, and Mn were determined by atomic absorption spectrometry (AAS, Hitachi Z-6000, Hitachi, Tokyo, Japan) of dry ash digested in HCl, P was measured spectrophotometrically (HP 8453, Hewlett Packard, CA, USA) in a HCl extract by the molybdate-hydrazine method (Halonen et al. 1983). All the nutrient analyses were performed on two seedlings per treatment and block, which were pooled and ground, and concentrations were measured in duplicate samples. The nutrient contents of the different plant parts were calculated by multiplying their dry mass by the respective nutrient concentrations. In addition, the B content was expressed as a proportion (B allocation) by dividing that of each part of the plant (roots, stems, and needles) by that of the whole plant.
Shoot water potential, soil water conditions and gas exchange
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Shoot water potential was measured with a Scholandertype pressure chamber (T. Pohja, Juupajoki, Finland) between 0900 and 1500 hours (lights came on at 0400 hours). The shoot was cut at the root collar, inserted into the pressure chamber and the water potential measured. After the measurement, the pot was sealed with Parafilm for subsequent determination of soil gravimetric water content (weighing the soil before and after drying at 105°C) and estimation of water potential, for which soil water retention curves were generated as described in Möttönen et al. (2001a). Gas exchange was measured with an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA) equipped with a CO2 injector (Li-Cor) between 0900 and 1500 hours (lights came on at 0400 hours). The apical tip and 4±1 cm of the shoot were enclosed in a conifer gas exchange chamber (LI-6400-05, Li-Cor). The measure-
Nitrogen uptake and its translocation to the needles were assessed by selecting five seedlings per treatment and block at random on day 134 (at the end of the drought period) and giving them 20 ml water within 30 min before feeding them with a 15N solution prepared from (NH4)2SO4 (N concentration 3.34 mmol dm−3) which contained 98% of its N in the form of 15N. The seedlings were watered with 10 ml of the solution and harvested 5 h after feeding. 15N was determined from the needles, which were separated from the stems and dried (at 60°C with forced ventilation for a minimum of 3 days), ground to a fine powder in a ball mill and subsequently analyzed for the N isotope ratio (δ15N). There were also 36 control seedlings (2 per treatment per block) to which 15N was not added, but which were otherwise watered similarly and the measurements performed in a similar way. The 15N analyses were carried out with a stable isotope analyzer
N experiment
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(Europa Scientific 20/20, PDZ Europa, Cheshire, UK) at the Waikato Stable Isotope Unit, University of Waikato, New Zealand. The results are expressed in the delta notation 15 N ¼ ½ðRsample =Rstandard Þ 1 1; 000 ð0=00Þ , where Rsample=15N/14N in the sample and Rstandard= 15 14 N/ N in the reference standard (N2 in air).
Statistical analysis Mean values for the measured seedlings per block and treatment were used in the analyses, as the individual seedlings were not true replicates (in the case of growth and nutrient data). Angular transformations were used for proportions. The data were subjected to an analysis of variance (ANOVA) using a model: block + B + D + B × D with the levels −B and +B for boron (B), and D0, D1 and D2 for drought (D). As theF-tests provided by routine analysis of variance reduce several comparisons to a single test (when df>1), more specific contrasts were also used. The F-tests for drought with two degrees of freedom were divided into two contrasts (c1 and c2), each with one degree of freedom [c1: D0 vs (D1 + D2)/2 and c2: D1 vs D2]. The soil water content, shoot water potential and gas exchange data, recorded on 7 days during the experiment, were analyzed using a repeated measures ANOVA model block + B + D + B × D, with day as the repeated factor and with the levels −B and +B for boron, and D0, D1 and D2 for drought. As only the interaction day × D was significant, these data were further subjected to an ANOVA and the contrasts were calculated as described above. All the statistical analyses were performed with SPSS (SPSS for Windows, Release 10.0.5, SPSS).
Results Growth, root tips, mycorrhizas and visible damage As the interactions B × D were non-significant (P≥0.381) when the growth, root tip and mycorrhiza data were analyzed, only the main effects (B and D) and contrasts (c1 and c2) are reported in Figs. 1 and 2. The height of the −B seedlings was on average 93% of that of the +B seedlings on day 134 (after drought) and 96% on day 142 (after recovery) (Fig. 1a). The main effect of B was significant only on day 142, however. In contrast, drought reduced height significantly on both measuring dates, so that the heights for D1 and D2 were 87% of the D0 figures on average on both measuring dates and significantly lower in D2 than D1 on day 134. No significant differences (P≥0.453) in root, stem, needle or total dry mass (Fig. 1b), total needle area (data not shown) or dry mass allocation (Fig. 1c) were observed between the two B treatments on day 134, but the root dry mass of the −B seedlings was 81% of that of the +B seedlings on day 142 (P=0.014) (Fig. 1b), and the +B
Fig. 1 a Mean stem height and means of b absolute and c proportional dry mass of seedlings on days 134 (after drought) and 142 (after recovery). Means of six replicates (total number of seedlings 144). Error bars give 95% confidence intervals for stem height and total dry mass. For levels of boron supply (−B and +B) and drought (D0, D1, and D2), see text. *, **, and *** stand for P≤0.05, P≤0.01, and P≤0.001, and ns for P>0.05. Interactions B × D non-significant, P≥0.381. Levels of D are compared using the contrastsc1 D0 vs (D1+D2)/2 and c2 D1 vs D2. In b and c; roots: cross-hatching, stems: solid white and needles:diagonal hatching
seedlings allocated proportionally more biomass to their roots than the −B seedlings (P=0.032) (Fig. 1c). Root, stem, and needle dry masses were significantly reduced by drought, and consequently the total dry mass in the D1 and D2 treatments was about 80% of that in D0 on both measuring dates (Fig. 1b). The +B seedlings had significantly more long roots (in absolute numbers and per unit root mass), mycorrhizas with a mantle (in absolute numbers, percentages and per unit root mass) and C. geophilum mycorrhizas (in absolute
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numbers, percentages and per unit root mass) and more mycorrhizas with a mantle (absolute numbers, percentages, and per unit root mass), and consequently more mycorrhizas altogether (in absolute numbers, percentages and per unit root mass) than the −B seedlings. The numbers of long root tips (absolute numbers), developing mycorrhizas (absolute numbers) and mycorrhizas with a mantle (absolute numbers and per unit root mass), and consequently the total number of root tips (absolute numbers and per unit root mass) and mycorrhizas (absolute numbers) were significantly lower in the D1 and D2 treatments than in D0 on day 134 (Fig. 2), and the numbers of long root tips (absolute numbers) and developing mycorrhizas (absolute numbers), and consequently the total number of root tips (absolute numbers), were significantly lower than in D0 on day 142. The difference between the 2 days in the number of root tips (absolute numbers and per unit root mass) was not affected by B or interaction between B and drought. However, the regeneration of root tips after rewatering seemed to occur only in D1 and tended to be more intensive in the −B seedlings than in the +B ones. Most of the seedlings showed no visible symptoms of damage in the upper shoot on day 134, but 14% of the −B seedlings in the D2 treatment were damaged (Fig. 3). The most severe damage class (abundant yellow or brown needles, dead apical tips) was present only in the −B seedlings in the D2 treatment. Soil water conditions, shoot water potential and gas exchange No significant differences (P≥0.115) in soil water conditions or gas exchange were observed between the two B treatments in repeated measures ANOVA or ANOVA at each measuring date (Fig. 4), but the shoot water potential Fig. 2 Mean numbers a of root tips, b of short root tips in different categories as percentages of all short root tips, and c of root tips per unit root dry mass in the seedlings on days 134 (after drought) and 142 (after recovery). Means of six replicates (total number of seedlings 72). Error bars give 95% confidence intervals for the total number of root tips and number of root tips per unit dry mass. For levels of boron supply (−B and+B) and drought (D0, D1, and D2), see text. For statistical comparisons, see Fig. 1. Interactions B × D non-significant, P≥0.128. Long roots:checked hatching (except in b, where long roots are excluded), non-mycorrhizal root tips: solid black, developing mycorrhizas: cross-hatching, mycorrhizas with a mantle: diagonal hatching, and Cenococcum geophilum: solid gray
numbers, percentages and per unit root mass), and consequently more root tips (in absolute numbers and per unit root mass) and mycorrhizas (mycorrhizas with a mantle and C. geophilum, in absolute numbers, percentages and per unit root mass) than the −B seedlings on day 134, but they had significantly fewer non-mycorrhizal root tips (in absolute numbers, percentages and per unit root mass) and developing mycorrhizas (in percentages) (Fig. 2). On day 142 the +B seedlings still had significantly fewer non-mycorrhizal root tips (absolute
Fig. 3 Visible damage to the upper shoot of the seedlings. For levels of boron (−Band +B) and drought (D0, D1, and D2), see text. For damage classification criteria, see text. The total number of seedlings was 1,152. Mild damage: solid white, medium damage: solid gray, and severe damage: solid black
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of the +B seedlings was lower than that of the −B seedlings on day 5 of the drought period (P=0.016) (Fig. 4b). The interactions day × B × D and B × D were non-significant (P≥0.158), but the interaction day × D was significant (P≤0.044) for all the variables (Fig. 4). The gravimetric soil water content was lower in the D1 and D2 treatments than in D0 (P<0.001) on all the measuring dates during the drought period (Fig. 4a), being about 12% (w/w) in D0, but decreasing to 2% by day 5 of the drought period in D1 and D2. These values correspond to a soil water potential above −0.01 MPa in D0 and below −1.0 MPa in D1 and D2. The effect of drought on gravimetric soil water content was also significant during the recovery period (P≤0.041), as this was lower, being about 12% (w/w) in the D1 and D2 treatments, than in D0, 14% (w/w) (P≤0.012). As expected, shoot water potential (Ψshoot) was lower in the D1 and D2 treatments than in D0 on all measuring dates during the drought period (P<0.001), and lower in D1 than in D2 on days 2, and 3 of drought (P<0.015) (Fig. 4b). In D0 it was −0.9 MPa by day 5 of drought, while in D1 and D2 it had decreased to −1.6 MPa. During the recovery period readings in the D1 and D2 treatments returned to close to the D0 level on the first day of recovery and were not affected by drought (P≥0.231). Net photosynthetic rate (A) was lower in the D1 and D2 treatments than in D0 (P<0.001) on all measuring dates during the drought periods (Fig. 4c). In D0 it was about 8.5 μmol m−2 s−1, while in D1 and D2 it decreased rapidly to 2 μmol m−2 s−1 by day 5 of drought. It was lower in D1 and D2 than in D0 (P<0.021) on all measuring dates during the recovery period as well, being about 6.0 μmol m−2 s−1 in D1 and D2 and 7.2 μmol m−2 s−1 in D0 on the last day of recovery. Moreover, it was lower in D1 than in D2 (P=0.016) on the first day of recovery. The changes in stomatal conductance (gs) were similar to those observed in A (Fig. 4d). It was lower in the D1 and D2 treatments than in D0 (P<0.001) on all measuring dates during the drought periods, and lower in D2 than in D1 (P≤0.001) on the first day of drought. On day 5 of drought it averaged 0.14 mol m−2 s−1 in D0, while in D1 and D2 it was under 0.02 mol m−2 s−1. It was lower in D1 and D2 than in D0 (P<0.001) on all measuring dates during the recovery period. Following the reduction in gs, declines in the intercellular CO2 concentration (Ci) were observed, this being lower in D1 and D2 than in D0 (P<0.001) on all measuring dates during drought, and lower in D2 than in D1 on days 1 and 2 of drought (P≤0.002) (Fig. 4e). It was about 250 μmol mol−1 in D0, but had decreased to 150 μmol mol−1 in D1 and D2 by day 5 of drought. It was also lower (P<0.001) in D1 and D2 after rewatering being about 200 μmol mol−1 versus 250 μmol mol−1 in D0. Nutrients The mean B concentrations in the roots, stems and needles of the −B seedlings on both measuring dates were
6 mg kg−1, 7 mg kg−1 and 6 mg kg−1, while the respective concentrations in the +B seedlings were 16 mg kg−1, 28 mg kg−1, and 34 mg kg−1 (Fig. 5), and consequently the total B content of the −B seedlings was 10 μg and that of the +B seedlings 46 μg (Fig. 6a), the main effect of B being significant. Drought significantly increased root B concentration and it was significantly higher in the D1 and D2 treatments than in D0 on day 134, but did not markedly affect B concentrations in the stems and needles (Fig. 5). In contrast, the total B content was significantly reduced by drought and was significantly lower in D1 and D2 than in D0 on days 134 and 142 (Fig. 6a), and significantly lower in D2 than in D1 on day 134. The +B seedlings allocated less B to the roots (P≤0.001) and more to the needles (P≤0.001) than the −B seedlings on day 134, less B to the roots (P≤0.001) and stems (P≤0.001) and consequently more to the needles (P≤0.001) than the –B seedlings on day 142 (Fig. 6b). The +B seedlings had significantly higher N, P, Ca and Mg concentrations and P, Ca and Mg contents in their needles than the –B seedlings on day 134, but not on day 142 (Fig. 7). In contrast, K and Mn concentrations and contents were not affected by B (data not shown). Needle N, Ca and Mg concentrations were significantly increased by drought when measured on day 134, so that N and Mg concentrations were significantly higher in the D1 and D2 treatments than in D0 and N and Ca concentrations were significantly higher in D2 than in D1. On day 142 only the N concentrations were significantly increased by drought, being significantly higher in D1 and D2 than in D0 and higher in D2 than in D1. Conversely, the P content was significantly higher in D0 than in D1 and D2 on day 142. 15
N experiment
The δ15N signature was 44‰ in the needles of the D0 seedlings, but 4‰ and 2‰ respectively in D1 and D2 on day 134. In the control seedlings (not supplied with 15N) it was about –4‰. B treatment did not significantly affect δ15N (P=0.094), but the effect of drought was significant (P≤0.001), as the D1 and D2 seedlings had significantly lower δ15N than the D0 ones (P≤0.001). The interaction B × D was non-significant (P=0.118).
Discussion Where the present needle B concentrations were 6 mg kg−1 in the −B seedlings and 34 mg kg−1 in the +B seedlings, concentrations <4–5 mg kg−1 are reported to induce shoot die-back in mature Norway spruces (Brække 1977, 1979; Silfverberg 1980; Aronsson 1983; Lipas 1990) and concentrations above 8 mg kg−1 have been considered adequate (Brække 1977, 1979; Aronsson 1983). The needle B concentrations in the +B seedlings were not yet even close to a toxic level. In 1-year old Norway spruce seedlings grown in the nursery the growth of the seedlings
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Fig. 4 Mean a soil gravimetric water content, b shoot water potential, c net photosynthetic rate per unit of projected needle area, d stomatal conductance per unit of projected needle area, and e intercellular CO2concentration. Means of six replicates (total
number of seedlings 36). For levels of boron (−B: black circle and +B: open circle) and drought (D0, D1, and D2), see text. The arrow indicates the start of rewatering
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Fig. 6 Means of a absolute and b proportional B content in different parts of the seedlings on days 134 (after drought) and 142 (after recovery). Means of six replicates (total number of seedlings 72). Error bars give 95% confidence intervals for the total content. For levels of boron supply (−B and +B) and drought (D0,D1, and D2), see text. For statistical comparisons, see Fig. 1. Interactions B × D non-significant, P≥0.116. Roots: cross-hatching, stems: solid white and needles:diagonal hatching
Fig. 5 Mean B concentrations in a roots, b stems, and c needles with 95% confidence intervals, on days 134 (after drought) and 142 (after recovery). Means of six replicates (total number of seedlings 72). For levels of boron supply (−B and +B) and drought (D0,D1, and D2), see text. For statistical comparisons, see Fig. 1. Interactions B × D non-significant, P≥0.268
started to decrease when needle B concentrations were over 100 mg kg−1(Rikala 2003). We found that low B reduced the numbers of root tips and mycorrhizas, as in our previous studies (Möttönen et al. 2001a,b). Boron promoted mycorrhiza development here so that the +B seedlings had fewer non-mycorrhizal root tips and developing mycorrhizas and more fully developed mycorrhizas. Non-mycorrhizal short roots and developing mycorrhizas typically occur in seedlings supplied with high doses of macronutrients (de la Rosa et al. 1998, 1999), and here an adequate B supply somewhat counteracted the negative effect of fertilization on mycorrhizas.
We found visible symptoms of damage in the upper shoot in the −B seedlings, especially in those exposed to two periods of drought, where the damage was severe and including dead apical tips. In contrast, (Möttönen et al. 2001a) did not observe any visible damage after one period of drought in Norway spruce seedlings which had equally low-B concentrations. In nursery seedlings of the loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm.) B concentrations as low as 1.9 mg kg−1 have been observed to induce damage to shoot tips and buds (Stone et al. 1982). After planting out, these damaged seedlings developed into bushy plants and showed abnormal height growth during the first 2 years (Stone et al. 1982). Correspondingly, the typical symptoms of B deficiency observed in Norway spruce are multiple leaders and repeated leader dieback, giving the tree a bushy appearance (Silfverberg 1980). The milder visible damage observed here (discoloration of needles) may further detract from growth. Although no interactions between B and drought were found in the growth of the present seedlings, the +B seedlings had better height growth and especially higher
221 Fig. 7 Mean needle nutrient concentrations and contents, with 95% confidence intervals, in the seedlings on days 134 (after drought) and 142 (after recovery). Means of six replicates (total number of seedlings 72). For levels of boron supply (−B and +B) and drought (D0, D1, and D2), see text. For statistical comparisons, see Fig. 1. Interactions B × D nonsignificant, P≥0.262
root biomass than the −B seedlings after recovery from drought. Actually, it has also been observed earlier that some tree species respond to drought with increases in root:shoot ratios (Joslin et al. 2000). As our +B seedlings had allocated more biomass to the roots than the −B seedlings especially after repeated drought during rewatering, it can be suggested that they may recover better from future droughts as well. In our earlier experiment the +B seedlings lost water more rapidly than the −B seedlings, leading to slightly
decreased Ψshoot, A and gs on the 4th day of drought (Möttönen et al. 2001a). The present +B seedlings also had slightly lower Ψshoot than the −B seedlings on the 5th day of drought, but A and gs were not significantly affected by B. This suggests a slightly more rapid water uptake of the +B seedlings which led to a more rapid water depletion during the first days of drought. Generally, A and gs were not markedly affected by B during the drought or after recovery, suggesting no direct effect of B on gas exchange. The seedlings with the most
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severe symptoms of visible damage were not included in the gas exchange measurements, however. This may have minimized any indirect effect of B on gas exchange. On the other hand, Yu et al. (2002) reported severe B deficiency to lower A in ramie (Boehmeria nivea L.), rape (Brassica napus L.) and cotton (Gossypium hirsutum L.). The lower A was related to a decrease in chlorophyll content in B-deficient plants. In addition, Zhao and Oosterhuis (2003) reported that a B deficiency in cotton considerably reduced A and also growth, although A and growth only began to differ between the +B and −B treatments after the first 2 weeks. The B concentration in the −B treatment was 20 mg kg−1 which indicates a severe deficiency for cotton (Zhao and Oosterhuis 2002), but above those critical B concentrations A and the growth of cotton in the field were not affected (Zhao and Oosterhuis 2002). It seems that A of plants is not affected by B unless the deficiency is severe and growth is also affected. Actually, no direct effect of B on A has been observed previously, either, but B deficiency may indirectly affect A by reducing the photosynthetic area and altering leaf constituents, e.g. stomatal frequency and aperture and the chlorophyll, soluble sugar and protein content of leaves (Dell and Huang 1997). Ψshoot, A, gs and Ci were all lowered by drought, the decrease in A being largely dependent on gs, as indicated by the reduced Ci. Stomatal regulation of A during drought has been well documented, as recently reviewed by Yordanov et al. (2000), Medrano et al. (2002), and Chaves et al. (2003), for example. At least under conditions of mild drought, the stomata often play the dominant role in controlling the decline in net CO2 uptake, by causing a decrease in Ci. It seems, however, that even though the seedlings suffered a drought severe enough to have a major effect on A and gs, these responses were not dependent on their B status. Ψshoot recovered almost entirely after rewatering, and an increase in gas exchange in the drought-treated plants after rewatering indicated in turn that the basic mechanism of photosynthetic biochemistry and photochemistry was not impaired during drought. Actually, the rapid but only partial recovery of A in the drought-treated seedlings after rewatering seemed to be due to stomatal limitation, since gs, in particular, remained lower in the drought-treated seedlings compared with the watered ones. This aftereffect on gs can be thought of partly as protecting the plants against rapid consumption of the new water supply and has been attributed to a persistent effect of abscisic acid produced during drought (Fischer et al. 1970). The nutrient status of the seedlings was good except for the low B concentrations in the −B seedlings (Jukka 1988; Brække 1994). Needle P, Ca, and Mg concentrations and contents were slightly higher in the +B seedlings than in the –B ones at the end of the drought probably partly due to the higher number of mycorrhizas. The higher needle P concentrations and contents may have also been due to higher root uptake of P due to a better function of root plasma membranes at adequate B, while an increased xylem Ca transport to the shoot at adequate B could be
suggested for a reason for increased needle Ca concentrations (Marschner 1995). None of the present seedlings received nutrient solution during the drought, whereas after recovery, during which time nutrients had again been supplied, no effects of the two B supply rates on nutrient concentrations or contents were detected. This suggests that under conditions of continuous adequate nutrient availability B has only a minor role in seedling nutrient status. Accordingly, the abundant N status of the seedlings may have been reflected in needle N concentrations, which were only slightly increased by B, and in 15N uptake and its translocation to the needles and N content, which were unaffected by the B treatments after drought. It seems that the −B seedlings were able to take up B at the early stages of the experiment but were not able to transport it up to the needles later in the drought period, due to reduced transpirational water flux. In addition, as the B concentrations were low in the −B seedlings, they were not able to retranslocate B from other plant parts either, although retranslocation of B has been observed in the Norway spruce (Lehto et al. 2000, 2004). The visible damage to the apical growth of the −B seedlings observed here after two drought periods may have been caused by this inadequate and/or non-continuous B supply to the continuously active meristematic areas, as also observed in the developing buds of mature Norway spruce trees growing in soil poor in B (S. Sutinen, personal communication). In the field, drought may reduce the availability of B and thus lower B and drought together may also reduce root growth, which in turn will detract from the already low-B uptake so that deficiencies develop. In summary, we found that low B reduced height growth, root biomass, formation of root tips and mycorrhizas and nutrient uptake in Norway spruce seedlings. In contrast, no direct effects of B on shoot water potential and gas exchange were observed. However, in the longer term low B may also affect shoot water potential and gas exchange. Repeated drought stress resulted in visible symptoms of damage to seedlings with low B, probably due to reduced B transport. These visible symptoms of damage seem to appear suddenly when the seedlings are exposed to drought. This finding can probably partly explain the suddenly occurring visible symptoms of B deficiency reported in the field studies. Acknowledgements We thank Ms. Leena Kuusisto, Ms. Maini Mononen, Ms. Rauni Oksman, and Ms. Maiju Tolvanen for their skillful technical assistance. We are also very grateful to Mr. Malcolm Hicks, for revising the language of this paper, and Dr. Sirkka Sutinen for comments on its content. This research was funded by the Academy of Finland (Decision no. 73515), the Maj and Tor Nessling Foundation, the Kemira Oy Foundation, and the Graduate School in Forest Sciences
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