Trees (1993) 8:31-38

�9 Springer-Verlag 1993

Growth and nutrition of small Betula pendula plants at different relative addition rates of iron

Anders G6ransson

Swedish University of Agricultural Sciences, Department of Ecology and Environmental Research, P. O. Box 7072, S-750 07 Uppsala, Sweden

Received February 10/April 7, 1993

Summary. Small birch plants (Betulapendula Roth.) were cultivated in a hydroponic spray solution where the relative addition rate of iron (RFe; g g-1 day-l), was the growth- controlling variable. All other elements were added in free access. An additional treatment was performed where all nutrients, including iron, were in free access (FA). The plants showed deficiency symptoms at steady-state growth and severe limitation of iron, Rye 0.05 and 0.10 day -1. There were few symptoms at RFe of 0.15 or above. Plant relative growth rate (RG; g g-1 day-l), equalled the relative rate of increase in iron supply, RFe. Internal iron concentra- tion of the plants ranged from 40 to 70 p.g g-1 dry weight (DW) over the range for which iron supply was limiting growth. At FA, the internal concentration was approxi- mately 200 ~tg g-I DW without further increase in RG, demonstrating that iron may be taken up in excess without affecting growth. Internal concentrations of macronutrients were stable at the different RFe, except for Ca and Mg in shoots which were higher at low iron supply. Uptake rates of iron, calculated per root growth rate (I.tmol g-1 root DW), were approximately twice as high at RFe 0.20 as at 0.05 day -1. The effect of iron limitation on dry matter allocation to leaves was small, with increases in the root fraction being largely at the expense of the stem. Leaf area ratio was constant regardless of RFe and the specific leaf area tended to increase with increasing iron limitation. Net assimilation rate decreased by a factor of 6 from free access to severe iron limitation, largely accounting for the differ- ences in plant RG.

Key words: Biomass and carbon allocation - Birch - Iron limitation - Nutrient proportions - Steady-state growth

Introduction

The content of soluble iron in soils is very low in compari- son with the total Fe content. It is available to plants in an

ionic form, mainly as Fe3+, or in chelated form in soils as Fe(III)-chelates (see Marschner 1986; Mengel and Kirkby 1987). In percolates from the A-horizon in a Swedish pod- sol, Tyler (1981) showed that total iron in lysimeters may range from approximately 35 to 350 pM, with typical high peaks in the autumn. Availability of iron may be restricted at high pH and "lime-induced" chlorosis and iron defi- ciency are well documented (Lindsay 1974).

There have been several attempts to establish the re- quirement of plants for iron since it was first recognised as an essential mineral nutrient element. From traditional cul- ture solution experiments, using e. g. Hoagland or modified Hoagland solutions, the required external iron concentra- tion for growth of healthy-looking plants was estimated as approximately 1-20 gM (Asher and Edwards 1983). Ep- stein (1972) summarised adequate internal iron amounts as approximately 100 btg g-1 dry weight. Many plant species may have concentrations well above this. Generally, the relationship between internal micronutrient concentration and plant growth is inconsistently described. Plant proper- ties may vary with time and negative correlations between growth and internal nutrient concentration have been re- ported (see Steenbjerg 1951; Hewitt 1956; Smith 1962; H~iubling et al. 1985). The quantitative requirement of iron for growth is thus described with low accuracy using such undefined expressions as "healthy", "well-looking" or "deficient" plants.

In contrast to experiments in which nutrients are added in initially well-defined external concentrations, Ingestad and co-workers (e.g. Ericsson 1981; Ingestad 1982; In- gestad and K~ihr 1985; Ericsson and Ingestad 1988) used relative addition rate of nutrients as the growth-controlling variable. By quantifying the added nutrient amount over time and adding it in relation to the expected plant require- ment, plants were at steadjc-state growth which is strictly mathematically defined (Agren 1985). The reliability of the interpretation of the collected data depends on the ability to maintain plants at steady-state growth. Carbon allocation, relative growth rate, and internal nutrient con- centrations are generally stable during the exponential phase of growth. The basic methodology and theory of the

32

relative nutrient supply concept have been discussed in detail by ~gren (1985), Ingestad and Lund (1986) and Ingestad and Agren (1988, 1992).

The aim of the present investigation was to study plant nutrition and partitioning of biomass using different relative addition rates of iron as the growth-controlling variable.

Materials and methods

Pre-treatment. Birch (Betula pendula Roth.) seeds, collected from a maternal clone at Stockholm, Sweden, were sown in Petri dishes on filter paper soaked with distilled water and germinated under continuous low light, =20 ~tmol m -2 s -1 (400-700 nm). After 10 days, approximately 200 seedlings were transferred to growth units (ingestad and Lund 1986) which were installed in climate chambers with artificial light (Osram HQIR 250 W/D), photon flux density =250~tmol m -2 s -1, (400- 700 nm), 24 h day; air and root temperature 20 ~ C and vapour pressure deficit (VPD) =0.6 MPa.

In the growth units, approximately 5 1 of distilled water was continu- ously circulated and sprayed on the roots at a flow rate of 10 1 per rain. The stock nutrient solution was added from burettes into the recirculating solution and the contents by weight in relation to nitrogen (N = 100) were: 65 K, 13 P, 7 Ca, 8.5 Mg, 9 S, 0.7 Fe, 0.4 Mn, 0.2 B, 0.06 Zn, 0.03 Cu and 0.007 Mo. Total N content in the stock solution was 2.5 g 1-1. All nutrients were added in free access (FA), to a constant pH of 3.9 - 4.1 and conductivity of approximately 100-200 ItS cm -1. The regulation of pH was carried out by using different proportions of NH4 + and NO3-. Conductivity (CDM Conductivity meter) and pH (priM63 digital pH meter, GK2501C combined pH electrodes; both Radiometer, Copen- hagen, Denmark) were automatically measured every 10 min and titra- tions were performed and controlled by a computer (IBM PC). To avoid chemical precipitation of iron, a low pH was used, resulting in a some- what lower maximal relative growth rate (RG) than at higher pH (Ingestad 1982).

After 1 -2 weeks, plant groups were harvested and analysed for iron. Of the remaining plants, 60 of uniform size were divided into groups of 4. Fresh weights were determined (=50 mg per plant) and their iron content was calculated from the content of the harvested plants. The plants were grown in an iron-free nutrient solution for 1 -2 weeks and the other nutrients were titrated as described above which resulted in a dilution of the internal iron concentration (cf. Ingestad and Lund 1986). Thereafter additions of iron, as iron nitrate, were performed in relation to the calculated content in the plants and at different limitation levels, i. e. at relative addition rate of iron (RFe) of 0.05, 0.10, 0.15 and 0.20 day -1 . At all values of RFe, the growth rates acclimated to the rate of addition. The period of adjustment lasted from a few days (near-maximum) to several weeks (severe iron limitation). To check that steady-state condi- tions were obtained, 5 plant groups were harvested at intervals during the period of adjustment and R6 estimated.

Experiments. When the plants were at steady-state nutrition, the fresh weights (FW) of 10 groups of 4 plants were each carefully weighed and put back into the units. Conductivity and pH were titrated as in the pre-treatments with the iron free nutrient solution to a constant value: 150 itS cm -1 and pH 3.9-4.1. Iron was added at the same relative rates as in the pretreatments. In an additional treatment, FA, all nutrient elements (including iron) were given at free access and the same pH. These plants attained a maximal Ro of approximately 0.22 day -1. Two replicates were made at each RFe.

Measurements. Three consecutive harvests of 5, 3 and 2 plant groups were performed during the experiments. Fresh and dry weights (DW; 65~ C for 2 days) of roots, stems and leaves as well as leaf area (Li-Cor M 3100 area meter, Li-Cor Inc., Lincoln, Neb., USA) were measured at each harvest. The leaf fraction was divided into three classes: newly developed, young fully developed, and old leaves. At each harvest, plant content of all macroelements and iron were analysed and, at the third and

last harvest, these analyses were made for stems, roots and leaves sepa- rately. Total nitrogen was determined by a micro-Kieldahl method using a flow injection analyser (Bifok FIA 5010, Tecator, H6gan~s, Sweden) with gas diffusion and using phenol red as indicator (Svensson and Anf~ilt 1982). The analysis of the other elements was performed by plasma atomic emission spectrometry (ICP-AES Instrumentation Labo- ratory IL P-200, Andover, Mass., USA or Jobin Yvon J 70+, Long- jumeau, France) after the samples had been digested for 2 days in a 2.5 : 1 (v/v) mixture of nitric acid and perchloric acid (BHD, Aristar). At the end of each experiment, samples were taken of the culture solution for analyses of all macronutrients and iron. The laboratory is linked to the IUFRO intercalibration network, Forestry Commission of N. S. W., Australia, for calibration purposes.

Non-structural carbohydrates were analysed as described in Steen and Larsson (1986) with small modifications (A. Flower-Ellis personal communication).

Calculations. The average relative growth rate, Ro (day -1) over the whole experimental period was calculated by fitting an exponential curve to weights (W) on different days (t),

W = Woe bt (1)

where b (= RG) is a constant. The net assimilation rate, NAR (kg DW m -2 day -1) was calculated from

Rc NAR = LAR (2)

where the leaf area ratio, LAR (m 2 kg -1 plant DW), is the product of the specific leaf area, SLA (m 2 kg -1 leaf DW), and leaf weight ratio LWR, (leaf DW plant -1 DW):

LAR = SLA x LWR (3)

The uptake rate of iron per unit root growth rate (gmol Fe g-1 root DW) was calculated as

dFe Fe W - x I ( 4 )

dWr W Wr

where Fe is the iron amount, W plant biomass and Wr root biomass.

Statistics. The statistical analyses were carried out by 1-way ANOVA (Statgraphics 1988). The pair-wise comparisons were done by the Least Significant Difference method at the confidence level 0.05.

0.20

0.15 .8

0.10

0 . 0 5

i i

0.(35 0.1'0 0.1'5 0.'P0 RFe, d a y -1

Fig. 1. The relationship between the relative addition rate of iron, RFe (g g-1 day 1 ), and the relative growth rate, R6 (g g-1 day 1), of small birch plants. The equation of the fitted line was y =-0.014+1.172x with r 2 = 0.98. The symbols represent the mean RG of each experiment, calcu- lated from four weight determinations

Resu l t s 75 ! Visual symptoms

During the pre-experiments and adjustment to steady-state 50 growth, the plants exhibited iron deficiency symptoms with interveinal chlorosis in younger leaves and green '~

25 healthy older leaves. When the plants were at steady-state 5-

"0

growth, symptoms typical of severe limitation became more pronounced. At severe limitation, RFe 0.05, all the "~ 0 leaves were yellow with white patches and necrotic spots *~ on the oldest. The roots were apricot-coloured and some of the shoots had reduced apical meristem growth, most typi- 25 cally in the RFe 0.05 treatment. With increasing RFe other symptoms developed, and all leaves were light green in the

50 0.10 treatment. There were no visual deficiency symptoms, or only minor ones, at a RFe of 0.15 or higher.

Growth and biomass partitioning

During the exponential phase of growth, which varied from approximately 25 (FA) to 100 days (0.05), the fresh weight of the individual plants increased from approximately

a . o . ~

"o o

m= b _=

Leaf

Stem

Root

33

0.05 0.10 0.15 0.20 FA RFe, day "1

Fig. 2. Dry matter allocation to leaf, stem and root (per cent of plant DW) at different relative addition rates of iron, RF~ (g g-1 day-l). Free access of all nutrient elements is denoted by FA and bars followed by the same letter within each fraction are not significantly different (LSD = 0.05). The calculations are based on three harvest occasions in each experiment

Table 1. Internal concentration of iron (gg g-i DW _+ SD) and nitrogen (mg g-1 DW _+ SD) at different relative addition rates of iron, RFe (g g-1 day -1) and the relative value of K, P, Ca, Mg S and Fe to N. In each experiment, analyses were performed for three consecutive harvests on whole plants from 5, 3 and 2 groups of 4 plants. Leaf, stem and root

fractions were analysed at final harvest on 2 groups of 4 plants. Means within each plant fraction and mineral nutrient, followed by different letters, are significantly different at the 0.05 level (LSD). The relative growth rate, RG (g g-1 day-l), was calculated by linear regression based on 4 weight determinations

RFe RG r 2 Concentrations

Fe N

Proportions

N K P Ca Mg S Fe

Plant 0.05 0.046_+0.005 >0.98 442-+ 2 0.10 0.098+_0.015 >0.99 43a-+ 4 0.15 0.171_+0.006 >0.99 60b_+ 2 0.20 0.217_+0.012 >0.99 69b_+ 6 FA 0.221_+0.003 >0.99 216~_+30

Leaf 0.05 0.049_+0.007 >0.98 29~_+ 4 0.10 0.101_+0.015 >0.99 25~+ 2 0.15 0.148_+0.026 >0.95 55b_+ 3 0.20 0.210_+0.005 >0.99 66b_+ 1 FA 0.220_+0.002 >0.99 193c_+23

S[em 0.05 0.059_+0.006 >0.99 272_+ 9 0.10 0.125_+0.024 >0.99 202+ 1 0.15 0.221_+0.001 >0.99 28~_+ 2 0.20 0.270_+0.010 >0.99 41a_+ 2 FA 0.223_+0.001 >0.99 1048_+26

Root 0.05 0.037_+0.002 >0.98 59a_+ 6 0.10 0.083_+0.014 >0.99 80a_+ 12 0.15 0.166_+0.003 >0.99 104a_+ 8 0.20 0.199_+0.001 >0.99 1082_+ 4 FA 0.221 _+ 0.001 >0.99 454b_+ 99

44.3a__+2.6 100 71 a 16 a 10 a 16 a 9 ab 0.10 ab 53.6c+2.3 100 70 a 16 a 9 b 12 b 9 a 0.08 a 49.7b_+0.2 100 58 b 16 a 8 be 12 b 9 ab 0.12 bc 49.8b_+ 1.5 100 58 b 16 a 7 c 12 b 10 c 0.14 r 46.0 a _+ 0.2 100 67 a 15 b 5 d 8 c 9 b 0.47 d

45.8 +3 .0 100 81 a 17 14 a 22 8 ab 0.06 ab 53.8 _+1.8 100 76 ab 17 11 ab 16 7 a 0.05 a 49.2 _+0.8 100 50 c 18 11 ab 16 8 a 0.11 bc 49.4 _+3.0 100 44 c 19 9 bc 15 9 b 0.13 c 47.3 _+ 1.3 100 52 bc 15 7 c 9 8 ab 0.41 o

26.2 +1.9 100 73 14 13 a 13 5 0.10 a 31.4 +4.3 100 93 14 l0 b 10 5 0.06 a 30.8 +1.3 100 84 15 8 c 10 6 0.09 ~ 31.0 > 0.1 100 84 15 7 c 10 6 0.13 a 29.7 +0.3 100 90 14 8 r 7 6 0.35 b

43.8a_+0.3 51.7c +0.7 57.8d__+ 1.3 56.80+0.3 48.1b_+0.6

100 65 a 15 3 4 11 a 0.13 a 100 75 ab 16 2 3 11 a 0.15 a 100 68 ab 15 2 4 10 ~ 0.18 a 100 77 b 15 2 5 14 b 0.19 a 100 94 c 14 2 6 14 b 0.94 b

r 2 is the coefficient of determination

34

, i , i

�9 - - 0

0.20 �9 . . . . . . . . . . . . o - -

/

.~ 0.15 t �9 " 0.10

0.05

0 I I I I

0 50 1 O0 150 200 Fe, pg g-1 DW Fig. 3. The dependence of RG (g g-I day-l) on the internal iron concentration, Fe (~tg g-l DW). The equation of the fitted line was y = ~0.158+0.005x with r 2 = 0.87. Filled symbols represent plants at sub-maximum relative growth rate and the open symbols, plants at FA of all nutrients. Each symbol represents the mean value from three consecutive harvests

250

200 150

T

100

50

I I1 m I I1 I11 1 i i HI 0.05 0.10 0.15

RFe, day -1

I I1 I11

0.20 l U HI

FA

Fig. 4. Internal iron concentrations, Fe (~tg g-1 DW) in different leaf classes at different relative addition rates of iron, RF~ (g g-] day-l), and at final harvest. The different leaf classes were: I newly developed, H young fully developed and I l l old. Free access to all nutrients is denoted by FA and bars followed by the same letter within each fraction are not signifi- cantly different (LSD = 0.05). Each bar represents the mean value of two analyses

50 mg initially to a maximum of 14 g at the last harvest. RG was approximately equal to RFe with a coefficient of deter- mination, r2 of 0.98 (Fig. 1). When calculated between the different harvests, RG was found to be stable at every specified RFe in each experiment, with a r 2 of 0.98 or better. The relative growth rate of the different plant parts was also stable at steady-state growth. RG of leaves and roots was equal to RFe while RG of the stem was somewhat higher in the 0.15 and 0.20 treatment (Table 1). The rela- tive growth rate of total leaf area responded in a similar way to that of leaf and root biomass to RFe (Table 2). There was a tendency towards greater leaf expansion per unit of carbon invested in leaf growth (SLA) at iron-limitation (Table 2). The dry weight/fresh weight ratio of plant parts was unaffected, except in stems where the ratio was higher in the RFe 0.05 and 0.10 treatments.

The stable values of dry matter allocation to root, stem and leaf fractions calculated as per cent DW of plants, is shown in Fig. 2. There were significant differences be-

tween the treatments for roots and stems. Allocation to roots increased with increasing iron limitation, while that to stems decreased. At FA, root biomass was approximate- ly 21% of plant biomass compared to 27% at RFe 0 . 2 0 , and 33% at RFe of 0.05. Biomass partitioning to the leaves was approximately constant, at 5 8 - 6 1 % of DW, regardless of treatment, except in the RFe 0.10 treatment, where the leaf proportion was about 53%. Leaf area ratio was largely independent of RFe (Table 2), whereas net assimilation rate was greater at higher RFe.

Nutrient and carbohydrate status

The highest iron concentrations were found in roots and the lowest in stems, and increased with increasing RG up to maximum RG (Table 1). The plant iron concentration was 44 ~tg g-1 DW at RG 0.05 and 69 p.g g-1 DW at RG 0.20 (Fig. 3). At FA, the iron concentration in plants was ap-

Table 2. Relative growth rate of total leaf area, RGa (m 2 m -2 day 1), leaf area ratio, LAR (m 2 kg -~ plant DW), net assimilation rate, NAR (10 -3) (kg D W m 2 day-l) and specific leaf area, SLA (m 2 k g -1 leaf DW), at different relative addition rates of iron. Means followed by different

letters are significantly different at the 0.05 level (LSD). Each value is based on 4 - 6 measurements. RGa was calculated by linear regression on 4 area determinations

RFe R~a r 2 LAR NAR SLA

0.05 0.058 + 0.013 >0.99 23 .29+ 4.90 1.87 a +0 .55 0.10 0.110---_0.016 >0.99 25 .08+2.31 3.68a +-0.52 0.15 0.157 +- 0.017 >0.99 23.87 +- 2.86 7.60 b +- 2.00 0.20 0.198 +- 0.004 >0.99 22.65 +- 1.44 10.09 b +- 2.36 FA 0.220 ___ 0.003 >0.99 21.93 +- 1.15 10.56 c +- 0.82

40.19 abc _+6.25 44.62 c + 3.43 41.34 bc -t-6.06 38.08 ab +_ 1.85 36.07 a +'1.44

r 2, coefficient o f determination FA, flee access to all nutrients

35

10.0

7.5 3=

6

5.0

2.5

L R L R L R L R L R

0.05 0.10 0.15 0.20 FA

RFe, day-1

Fig. 5. Total non-structural carbohydrate percentage (whole bars) and starch percentage (filled bars) of total DW in leaves (L) and roots (R) of small birch plants at different relative addition rates of iron, Rr~ (g g-1 day-l). Free access to all nutrients is denoted by FA and bars followed by the same letter within each fraction are not significantly different (LSD -- 0.05). Each bar represents the mean value of two ana- lyses

proportion N : F e in plants was lower at FA (100:0.5) than at RFe 0.20 (100:0.14) and at RFe 0.05 (100:0.10). In iron-limited plants, the differences in iron concentration in the different leaf-age classes were small (Fig. 4) while, at FA, the older leaves were higher in iron than younger ones.

The uptake rate of iron calculated per unit root growth rate (dFe/dWr) increased with decreasing iron limitation (Table 3). At FA, the iron uptake rate per root unit growth rate was 3.9 times higher than at RFe 0.20. In three experi- ments, the external iron concentration in the culture solu- tion was below the analytical limit, otherwise it ranged between 18 to 360 nM (Table 4) at the end of the experi- ments.

In roots and leaves the non-structural carbohydrates (starch, fructose, glucose and sucrose) decreased with in- creasing iron-limitation (Fig. 5). Stored carbohydrate in leaves (starch concentration) was approximately 0.8% of DW at severe and medium limitation and 4.9% at moderate or no limitation. Starch in roots was less than 0.4% of DW in all treatments (Fig. 5).

D i s c u s s i o n

Table 3. Uptake rates of iron calculated as uptake rate per unit root growth rate, dFe/dWr (pmol g-I root DW). Means followed by different letters are significantly different at the 0.05 level (LSD). Each value is based on 6 measurements

RFe dFe/dWr

0.05 2.4 a --+0.7 0.10 2.5 a -+0.4 0.15 3.1 ab -+1.0 0.20 4.7 b -+0.8 FA 18.5 c -+2.6

Table 4. The range of element concentrations found in the culture solu- tion in the different treatments at final harvest. The concentrations are given as mM and for iron in nM

RFe NH4 + NO3- K P Ca Mg S Fe

0.05 1.46- 0.63- 1.82- 0.18- 0.08- 0.03- 0.27- * 1.71 0.74 2.26 0 . 3 0 0.09 0.03 0.29 18

0.10 0.01- 0.02- 0.01- 0.04- 0.02- 0.01- 0.07- * 1.34 0.85 3.12 0.36 0.18 0.13 0.37 180

0.15 0.89- 0.72- 2.52- 0.33- 0.15- 0.07- 0.31- * 2.19 1.60 3.91 0.33 0.34 0.27 0.48 360

0.20 0.89- 0.85- 3.07- 0.27- 0.19- 0.18- 0.25- 360- 1.16 0.96 3.84 0.27 0.25 0.27 0.26 360

FA 0.0IN- 0.15- 0.37- 0.01- 0.02- 0.14- 0.01- 72- 0.29 0.18 0.39 0.01 0.07 0.23 0.01 220

*, values below the analytical limit, approximately 9 x 10 -11 M

proximately 3 times higher than at RG 0.20. The internal proportions of the macronutrients in the plants (Table 1) were approximately stable, with increasing iron limitation, except for calcium and magnesium, which increased. The

There was a large effect of the rate of iron supply on plant growth and increase in RG was associated with a change in internal iron concentration from approximately 40 gg g-1 DW at severe iron limitation to 70 at maximum growth. The strong correlation between the controlling growth variable, RFe, and plant RG (Fig. 1) was also demonstrated in leaves and roots, while in stems the relative growth rate was higher, especially in the RVe 0.15 and 0.20 treatments (Table 1). However, because the stem constituted such a small fraction of plant weight, the higher RG value of the stem did not upset the expected relationship between RG of the plant and RVe. These results are in agreement with findings of Ingestad (1979), Ericsson and Ingestad (1988) and Ericsson and KNar (1993) in experiments in which the relative addition rates of nitrogen, phosphorus or potassi- um, respectively, were the controlling growth variable. A further increase in internal iron, up to 200 gg g-1 DW at free access, was not immediately utilised in plant biomass production but stored, probably in the form of phytoferritin (Seckbach 1972, 1982).

The intercept of the regression line on the abscissa (Fig. 3), may indicate that small amounts of iron are re- quired as structural iron or in trigger mechanisms. Similar results were found in grapevine varieties by H~iubling et al. (1985) and in three iron-limited Populus clones by Keller and Koch (1962) who concluded that chlorophyll synthesis did not occur until other iron requirements had been ful- filled. It may also be the case that at very low internal iron concentration, carbon uptake through photosynthesis is equal to carbon loss through respiration, resulting in an iron "compensation point" for growth. Changes in visual appearance and RG may then proceed when internal iron increases. The analysed content of iron at FA is in agree- ment with that normally found in leaves and needles of forest trees (e. g. Tamm 1964; Zasoski et al. 1990).

A negative correlation between internal micronutrient status of plants and growth, the Piper-Steenbjerg effect, has

36

often been reported (Steenbjerg 1951; Hewitt 1956; Smith 1962). Several suggestions to explain the effect have been made, e.g. variation in tissue age (Bates 1971) or transit- cation within plants (Loneragan 1978), but there is no consistent physiological explanation for why a small appli- cation of a limiting element to plants growing under condi- tions of extreme limitation may be followed by a decrease in internal concentration. The results of the present inves- tigation indicate a linear relationship (Fig. 3) between in- ternal iron concentration of the plant and the relative growth rate when RFe is the growth-controlling variable. These observations are not supportive of the Piper-Steen- bjerg effect. It has yet to be shown that the effect is not simply attributable to methodology at non steady-state growth, where nutrient demands are increasing, and where the internal iron status then depends on the sampling occa- sion (cf. Agren 1985; Ingestad and Agren 1992).

Iron in leaf tissues is generally considered to be firmly bound and not readily retranslocated within leaves or be- tween leaves of different ages (Brown 1961; Hocking and Pate 1978). Eventually, at steady-state growth in the pre- sent study, and regardless of age at limiting RFe, all leaves within a treatment had stable and similar internal iron concentrations which may be referred to as physiologically active (Fig. 4). At FA of iron, an accumulation over time was recorded, resulting in approximately 50% more iron in old than in young leaves. Such stored iron, probably not actively used in physiological processes, may be retranslo- cated in conditions of iron shortage, for example during adjustment to limiting iron-supply. It may be concluded that iron in excess of immediate demand may be translo- cated readily to growing meristems. In contrast to experi- ments with nitrogen or phosphorus limitation (Ingestad 1979; Ingestad and Kahr 1985; Ericsson and Ingestad 1988) in which the visual deficiency symptoms disap- peared regardless of limitation level, the Fe deficiency symptoms remained at severe iron shortage.

Small systematic changes were found in macronutrient proportions at the different iron supply rates (Table 1). Physiologically, such changes may be difficult to interpret but they may be linked to the plant utilisation of different elements for growth. Ericsson and Ingestad (1988) showed that nitrogen uptake was dependent on the uptake of phos- phorus, but no such link between the uptake of iron and nitrogen was found in the present investigation. The N: Fe ratio in the nutrient solution (100:0.7) as proposed by Ingestad (1979) may therefore be increased by a factor of 5 for small birch plants without affecting maximum RG (Table 1).

There are few reports on the effects of iron limitation on morphological characters in forest trees, but there seems to be a resemblance to iron limitation in agricultural crops (Brown 1961; Wallihan 1966; Brown and Jones 1977; Rtmheld and Marschner 1981). At severe iron limitation, RFe 0.05, dry matter partitioning to roots was significantly higher than at FA (Fig. 2) where the root proportion, 0.2 of plant DW, was the same as that found by Ingestad (1979) and Ericsson and Ingestad (1988) at FA. However, these authors reported larger increases in allocation to roots at severe nitrogen or phosphorus limitation, (RN or Rp = 0.05), than were found here for iron. Instead of the

long exploratory root systems that the plants developed at nitrogen or phosphorus limitation, a denser system with shorter roots was developed when limited by iron (data not shown). By growing the plants at steady-state nutrition, it was possible to calculate iron uptake rates with respect to root growth rates as described in Eq. 4. Apparently, dFe/dWr was less at iron limitation than at higher supply rates (Table 3). These uptake rates were found at extemal concentrations 1-2 orders of magnitudes lower than re- ported earlier (Asher and Edwards 1983). The range in dFe/dWr (excluding FA) was less than the range of plant RG. This may imply that root growth response (in terms of dry matter allocation) is an important component for the further acquisition of iron. However, to interpret this obser- vation further in terms of possible acclimation of root structure and function to iron supply would require infor- mation on the development of root length and the occur- rence of uptake sites for iron on the root surface (cf. Clark- son and Sanderson 1978).

Some information on whether structural or functional development of leaves is most affected by a decrease in iron supply may be gained from considering the relative growth rate in terms of its components of LAR and NAR. The effect of limited iron supply on LAR was small (Table 2) and the relative growth rates of both leaf biomass and total leaf area (RGa) were close to RFe (cf. Tables 1, 2). At RFe 0.05, NAR was only 18% of that at near maximum of free access of all nutrients. The difference in NAR at RFe 0.05 compared with that at RFe 0.20 was much greater than the difference in NAR where nitrogen, phosphorus or po- tassium was the limiting nutrient over the same range of supply (McDonald et al. 1991). Although carbon fixation as such was not measured, the marked reaction to iron limitation, with characteristically yellow and white or light green leaves, may have been attributable to low chlorophyll content (Keller and Koch 1962; Terry 1980; Terry and Low 1982) followed by decrease in photosynthe- sis (Bottrill et al. 1970; Nfitr 1972, 1975) and carbohydrate production. This assumption is supported by the finding of low non-structural carbohydrate concentrations at low RFe (Fig. 5).

Allocation of carbohydrates may be explained in terms of source-sink relations, where young, not fully-developed leaves and roots act as sinks. In experiments with small Betula plants in which the relative addition rate of nitrogen was the growth-controlling variable, McDonald et al. (1986) found higher starch contents in leaves at greater nitrogen limitation. The lower allocation of dry matter to roots at severe iron stress RFe 0.05 compared with that at severe nitrogen limitation may have been attributable to low carbohydrate availability for root growth. However, it is not known if low carbohydrate contents as such were actually limiting to growth at low RFe. It is possible that amounts of carbohydrate had acclimated to the RFe supply such that they were neither limiting to structural growth nor present in such amounts as to be accumulated in excess of current usage in structural growth. Apart from at ex- treme iron limitation RFe 0.05, dry matter allocation be- tween plant organs (Fig. 2) was similar to that which has been reported for nitrogen, phosphorus and sulphur (cf. Ericsson et al. 1992). However, the amount of leaf area

37

produced for a given investment of dry matter in leaf growth (SLA, Table 2) was very much greater at severe iron limitation than at severe nitrogen limitation. Ap- parently, area growth was less restricted by limited iron availability than by limited supply of nitrogen. Why this should be the case remains unclear. However, any advan- tage to plant growth rate that a high value of total leaf area per plant dry weight (LAR, Table 2) might have conferred, was offset by the much lower value of NAR at severe iron limitation that at equivalent nitrogen supply. The lower value of NAR was presumably attributable to lower values of net photosynthesis per leaf area (cf. Terry 1980). Thus, plant response in acclimating growth rate to nutrient supply is quite different where iron is the limiting nutrient than where nitrogen is most limiting.

Conclusions

Over a range of supply rates, plant growth rate was control- led by the availability of iron. The relationship between relative growth rate and internal iron concentration was approximately linear at sub-maximum relative addition rates of iron. At free access to all nutrients, the plant iron concentration was about 3 times larger than required for maximum growth. The commonly reported, negative rela- tionship between growth and internal iron or micronutrient concentration was not found in the present study. Dry matter allocation to leaves and leaf area ratio were both largely unaffected by iron supply. Specific leaf area was at least as high at iron limitation as at free access and maxi- mum growth but net assimilation rate was much lower at limiting iron supplies. It is concluded that acclimation of growth rate in Betula to decreased iron availability does not involve large changes in dry matter partitioning to leaves and leaf expansion but is primarily associated with decreased net assimilation rate.

Acknowledgements. The author is greatly indebted to Profs. T. Ingestad, S. Linder, G. I. ~gren, Drs. T. Ericsson and A. J. S. McDonald for constructive criticism and advice during the investigation. Ms. E. Ar- widsson, A. Carlsson, A. Flower-Ellis, A. Hovberg, Mr. R. Reutlert and L. Ostberg helped with technical assistance. Dr. M. K~ihr drew the figures. The investigation was supported by grants from the Swedish Council for Forestry and Agricultural Research.

References

/~gren GI (1985) Theory for growth of plants derived from the nitrogen productivity concept. Physiol Plant 64:17-28

Asher C J, Edwards DG (1983) Modern solution culture techniques. In: L~iuchli A, Bieleski RL (eds) Encyclopaedia of plant physiology, vol 15A. Springer, Berlin Heidelberg New York, pp 94- 119

Bates TE (1971) Factors affecting critical nutrient concentrations in plants and their evaluation: a review. Soil Sci 112:116- 130

Bottrill DE, Possingham JV, Kriedemann PE (1970) The effect of nutrient deficiencies on photosynthesis and respiration in spinach. Plant Soil 32:424-438

Brown JC (1961) Iron chlorosis in plants. Adv Agr 13:329-369 Brown JC, Jones WE (1977) Fitting plants nutritionally to soils. III.

Sorghum. Agr J 69:410-414

Clarkson DT, Sanderson J (1978) Sites of absorption and translocation of iron in barley roots. Plant Physiol 61:731 -736

Epstein E (1972) Mineral nutrition of plants: principles and perspectives. Wiley, New York

Ericsson T (1981) Effects of varied nitrogen stress on growth and nutri- tion in three Salix clones. Physiol Plant 51:423 -429

Ericsson T, Ingestad T (1988) Nutrition and growth of birch seedlings at varied relative phosphorus addition rates. Physiol Plant 72: 227-235

Ericsson T, Rytter L, Linder S (1992) Nutritional dynamics and require- ments of short rotation forests. In: Mitchell CP, Ford-Robertson JB, Hinckley T, Sennerby-Forsse L (ed) Ecophysiotogy of short rotation forest crops. Elsevier London, pp 35-65

Ericsson T, Kahr M (1993) Growth and nutrition of birch seedlings in relation to potassium supply rate. Trees 7: 78 - 85

H~ubling M, Rtmheld V, Marschner H (1985) Beziehungen zwischen Chlorosegrad, Eisengehalten und Blattwachstum von Weinreben auf verschiedenen Standorten. Vitis 24: 158-168

Hewitt EJ (1957) Some aspects of the relationships of nutrient supply to nutrient uptake and growth of plants as revealed from nutrient culture experiments. In: T Wallace (ed) Plant analysis and fertilizer prob- lems, Institut de Recherches pour les Huiles et O16agineux, Paris, pp 104-118

Hocking PJ, Pate JS (1978) Accumulation and distribution of mineral elements in the annual lupins Lupinus albus L. and Lupinus an- gustifolius L. Aust J Agric Res 29: 267-280

Ingestad T (1979) Nitrogen stress in birch seedlings. II. N, K, P, Ca and Mg nutrition. Physiol Plant 45: 149-157

Ingestad T (1982) Relative addition and external concentration; driving variables used in plant nutrition research. Plant Cell Environ 5: 443 -453

Ingestad T,/~gren GI (1988) Nutrient uptake and allocation at steady- state nutrition. Physiol Plant 72:450-459

Ingestad T, Agren GI (1992) Theories and methods on plant nutrition and growth. Physiol Plant 84: 177-184

Ingestad T, KLlar M (1985) Nutrition and growth of coniferous seedlings at varied relative nitrogen addition rate. Physiol Plant 65: 109-116

Ingestad T, Lund A-B (1986) Theory and techniques for steady state mineral nutrition and growth of plants. Scand J For Res 1: 439-453

Keller T, Koch W (1962) Der Einflug der MineralstoffernNu'ung auf CO2-gaswechsel und Blattpigmentgehalt der Pappel. Mitt Schweiz Anst Forstl Versuchswes 38: 283- 318

Lindsay WL (1974) Role of chelation in micro-nutrient availability. In: Carson EW (ed) The plant root and its environment. University Press of Virginia, Charlottesville, pp 507-524

Loneragan JF (1978) Anomalies in the relationship of nutrient concentra- tions to plant yield. In: Ferguson AR, Bieleski RL, Ferguson IB (eds) Proceedings 8th International colloquium on plant analysis and fertil- izer problems, NZ DSIR Auckland, Information Series No 134. Government Printer, Wellington, pp 283- 298

Marschner H (1986) Mineral nutrition of higher plants. Academic Press, London

Mengel K, Kirkby EA (1987) Principles of plant nutrition. Int Potash Inst., Bern

McDonald AJS, Lohammar T, Ericsson A (1986) Uptake of carbon and nitrogen at decreased nutrient availability in small birch (Betula pendula Roth.) plants. Tree Physiol 2:61 - 71

McDonald AJS, Ericsson T, Ingestad T (1991) Growth and nutrition of tree seedlings. In: Raghavendra AS (ed) Physiology of trees. Wiley, New York, pp 199-220

N&r L (1972) Influence of mineral nutrients on photosynthesis of higher plants. Photosynthetica 6 :80-99

N~itr L (1975) Influence of mineral nutrition on photosynthesis and the use of assimilates. In: Cooper JP (ed) Photosynthesis and productiv- ity in different environments, Cambridge University Press, pp 537 -554

ROmheld V, Marschner H (1981) Rhythmic iron stress reactions in sunflower at suboptimal iron supply. Physiol Plant 53: 347-353

Seckbach J (1972) Electron microscopical observations of leaf ferritin from iron-treated Xantium plants: localization and diversity in the organelle. J Ultrastruct Res 39:65 -76

38

Seckbach J (1982) Ferreting out the secrets of plant ferretin - a review. J Plant Nutr 54 :369-394

Smith PF (1962) Mineral analysis of plant tissues. Annu Rev Plant Physiol 13:81 - 108

Statgraphics (1988) Statistical graphics system by statistical graphics corporation. STSC, Rockville

Steen E, Larsson K (1986) Carbohydrates in roots and rhizomes of grasses. New Phytol 104:339-346

Steenbjerg F (1951 ) Yield curves and chemical plant analyses. Plant Soil 3: 97 -109

Svensson G, Anf~ilt T (1982) Rapid determination of ammonia in whole blood and plasma using flow injection analysis. Clin Chim Acta 119: 7 - 1 4

Tamm CO (1964) Determination of nutrient requirements of forest stands. Int Rev For Res 1 :115-170

Terry N (1980) Limiting factors in photosynthesis. I. Use of iron stress to control photochemical capacity in vivo. Plant Physio165:114-120

Terry N, Low G (1982) Leaf chlorophyll content and its relation to the intracellular localization of iron. J Plant Nutr 5:301 - 310

Tyler G (1981) Leaching of metals from the A-horizon of a spruce forest soil. Water Air Soil Pollut 15:353 -369

Wallihan EF (1966) Iron. In: Chapman HD (ed) Diagnostic criteria for plants and soils. Riverside, Calif., pp 203- 212

Zasoski RJ, Porada HJ, Ryan PJ, Greenleaf-Jenkins J, Gessel SP (1990) Observations of copper, zinc, iron and manganese status in western Washington forests. For Ecol Manage 37:7 -25