Nm Phytol.

(1984) 97, 000-000

IRON REQUIREMENTS O'F Cg AND Q PLANTS BY G. S. S M I T H , I. S. CORNFORTH AND H. V. HENDERSON Ruakura Soil and Plant Research Station, Ministry of Agriculture and Fisheries, Private Bag, Hamilton, New Zedland {Accepted 10 April 1984)
SUMMARY Plants having the C,i photosynthetic pathway required higher concentrations of iron in the nutrient solution for maximum growth when grown in sand culture than those wdth the C^ pathway. There were only small differences among species in the concentration of iron required in the leaves to achieve near maximum dry matter yield. This suggests that C3 and C4 plants differ in their ability to absorb iron from the root zone. Higher concentrations of iron were required in the nutrient solution for maximum growth of both the C3 and C, plants when nitrate, rather than ammonium nitrate, was the sole source of nitrogen. It seems likely that when nitrate was used, there was a decrease in availability of iron to the plant as a result of the increased alkalinity in the root zone. By contrast, the root zone was acidified when ammonium nitrate was applied. Iron concentrations greater than 2 to 5 /tg Fe ml~' in the nutrient solution reduced the dry matter yieWs of a number of plants grown with ammonium nitrate as the nitrogen source, probably because of a decrease in the absorption of phosphorus by the roots. Key words: C3 plants, Cj plants, iron requirements, nitrate, ammonium nitrate, phosphorus uptake, root zone pH. INTRODUCTION

In a recent investigation of the uptake by pasture plants grown in sand culture of tretnorgenic mycotoxins produced by Penicillium spp. (White et al., 1980). It was observed that C4 species readily became chlorotic. The chlorosis could not be attributed to the mycotoxins and was only alleviated when the plants were sprayed with 1 % ferrous sulphate. This response was unexpected because the concentration of iron (3 ptg Fe ml"-' as ferric citrate) in the nutrient solution applied to the plants was greater than that commonly used in other sand culture experiments (Hewitt, 1966). While there are many reports of plants developing iron chlorosis when grown in sand or water culture ((Hageman et aL, 1961; Hewitt, 1966; Brooking, 1976; Asher, 1977), and of differences among plant species and cultivars in susceptibility to iron deficiency (Weiss, 1943; Vose, 1963; Brown et al., 1972; Marschner, Romheld & Azarabadi, 1978), there appears to have been no systematic comparison of Cg and C^ plants. This paper examines difTerences in the iron requirements of a number of C3 and C^ plants.
MATERIALS AND M E T H O D S

Plant culture The Cg and C^ species listed in Table 1 were grown separately in plastic pots containing 1 kg of silica sand which had been treated with steam heated 18% hydrochloric acid plus 1 % oxalic acid in a ' Keebush' sand purification unit and
0028-646X/84/0S0543 +14 S03.00/0
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Table 1. Plant species used in the experiment

C3 species Grasses

Browntop Cocksfoot Perennial ryegrass

Legumes

Agrostis capillaris L. {A. tenuis Sibth.) Dactylis glomerata L. cv. Grasslands .\panui Lolium perenne L. cv. Grasslands Nu[ Medicago sativa L. cv. Wairau TrifoUum pratense L. cv. Grasslands Turoa Trifohum repens L. cv. Grassland Pitau Pennisetum clmidestinum Hochst. ex Chiou Zea mays L. cv. Pioneer Paspalum dilatatum Poir. cv. Grasslands 15 Sorghum bicolor (L.) Moench

Lucerne Red clover White clover

C, species

Kikuyu Maize Paspalum Sorghum

then washed with deionized water (Hewitt, 1966). Two seeds of maize and sorghum were sown per pot, while ISO seeds of all other species were sown per pot. The experiment was carried out in a glasshouse with a mean daily air temperature of 22 C (IS C m^in to 27 C max). Mercury-vapour lamps (Philips HLRG) giving a photosynthetically active radiation of 400 /tmol m^^ s~S were used tO' extend the length of the day to 16 h. Treatments and nutrient solutions Iron was applied as ferric citrate at concentrations of 0-312, 0-6S2, 1-25, 2-5, S-0 and 10-0/<g Fe ml~^ either in a nutrient solution hased on ammonium nitrate or on nitrate alone (Table 2). Analytical grade chemicals were used to make up the nutrient solutions.. The initial pH of the solutions was 6-0 in both cases. Nutrient solutions were applied to the plants at a rate of 50 ml per pot three times a week.. Moisture stress was avoided by adding deionized water to the sand to maintain it at 7S % of saturation capacity. Four replicates of each treatment were set up in a randomized block design. Measurements Shoots and roots were harvested 60 d after seed germination. Chemical analyses were carried out on the most recent fully expanded leaf for maize and sorghum, and on whole shoots for the other species. As there is a close correlation between iron and chlorophyll contents (Jacobson & Oertli, 1956; DeKock et al., 1960), chlorophyll was extracted from 0-1 g of freshly harvested leaf tissue in cold 80 % (V/V) aqueous acetone until all pigment was removed. The extract was centrifuged to remove particulate material and the absorbance of the supernatent was measured at 649, 654 and 663 nm. The chlorophyll a and b contents were calculated by the nnethod of Bruinsma (1963). The reniaining shoot and root tissue was dried at 60 C overnight, weighed and analysed for macro and micro elements by the procedures summarized by Smith, Middleton & Edmonds (1980). Total iron was deternained by atomic absorption spectroscopy; ' active iron' was extracted from 0-5 g dry tissue with 15 ml of ether saturated 0-1 M HCl for 20 min at room temperature (BoMe-Jones, 19S5). The pH of the root zone was determined on days 20, 40 and 60,. The

Iron requirements of C3 and C^ plants

545

Table 2. Concentration of macro and microeleinents {fig ml^^) in the ammonium nitrate{A) and nitrate-only (B) nutrient solution before application to plants
Macroelements A NH.,-I\" NO,-N P S 133 133 25 35 218 198 26 56 6 B JVIacroelements A B 0-5 0-04 3-0 0-5 0-01 0-25

B
265 26 35 72 198 26 336 6 Cu Fe .Mn Mo Zn

0-5 0-04 3-0 0-5 0-01 0-25

Mg Ca Na

measurements were made on 20 ml of solution displaced from around the roots. Preliminary experiments had shown that up to SO ml of solution could be displaced hy applying deionized water to the surface of the sand before it was diluted. Statistical analysis Analyses of variance were carried out on the variables within each species presented in the tables. Between species, or photosynthetic pathway group, analyses were performed on summary results calculated for each species. The relationship between dry matter yield ( Yd) and iron concentration in the nutrient solution {x), and between iron concentration in the leaf ( YL) and iron concentration in the nutrient solution (x) was approximated by an inverse polynonnial. Yd or YL = xl{l+bx + cx') with parameters a, b, and c. Use of this function has advantages over ordinary quadratic polynomials in that it is non-negative, bounded, has no built-in syoimetry about the optimum and has an asymptote when c = 0. The relationship between iron concentrations in the roots and iron concentrations in the nutrient solution was described by a straight-line relationship on log transformed data.

RESULTS

Effect of nitrogen source and plant species on pH in the root zone

Plants grown on tbe ammonium nitrate-based nutrient solution reduced the pH from 6-0 to between 4-1 and 5-2, while those grown on the nitrate only based solution increased the pH from 6-0 to between 7-0 and 7-5 (Table 3). By contrast there were only small differences among tbe plant species. For example, when the CJ plants were grown on the ammonium nitrate solution the pH of the root zone was reduced to a slightly lower range of between 4-1 and 4-4 compared to 4-4 and 5-2 for the C3 species. Increasing the iron concentration in the nutrient solution had no significant effect on tbe pH in the root zone nor did it interact with nitrogen source or plant species.
19-2

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Table 3. Effect of nitrogen source and plant species on the pH in the root zone
Nitrogen source Plant species Cg species Browntop Cocksfoot Perenniai n^^grass Lucerne Red clover White clover C4 species Kikuyu Maize Paspalum Sorghum Mean Cg grasses Mean Cj legumes Mean C^ species Ammonium nitrate Nitrate only

4-g 4-7 4.4 4-6 5-2 4-9

4-]

7.4 7-5 7-5 7-0 7-1 7-2 74 7'3 7'1 7'5 7-5 7-1 7-3 0-2

4.4 4-3 4-1 4-6 4.9 4-2 0-3

LSD (5%)

Effect on dry matter yields

The iron requirements of the C^ species were greater than those of the Cg plants irrespective of the nitrogen source (Figs 1, 2). For example, maximum growth of the C3 plants grown on the nitrate-based solution occurred when the iron concentration in the solution ranged from 2 to 5 /tg Fe ml~^, but for the Cj species, dry matter yields were still increasing with iron concentrations of up to 10 /ig Fe nnl~^ in the solution. Sitnilarly, when nitrogen was supplied as ammonium nitrate the iron requirements of the Cg and Cj plants v?ere between 1 to 2-5 fig Fe ml^^ and 2-5 to 3-0 fig Fe ml"'',, respectively. In general, dry matter yields of all plants were greater when nitrogen was provided as nitrate (Figs 1, 2). Moreover, a higher concentration of iron in the nutrient solution was required byplants for maximum growth when grown on the nitrate-based solution then on the ammonium nitrate-based solution. Growth of a number of plants was also adversely afFected by iron. Dry matter yields of browntop, cocksfoot, and to a lesser extent of kikuyu, maize, ryegrass and sorghum were reduced by iron concentrations greater than 2 to 5 /ig Fe ml"^ in solution, particularly when the plants were grown on the ammonium nitrate-based solution.
Chemical composition

Chlorophyll. All species were chlorotic when grown on the lowest iron concentration, the severity of the disorder being reflected in the chlorophyll concentration of the shoots (Table 4). Chlorosis was generally greater when all the nitrogen was provided as nitrate and in those species which were particularly sensitive to low concentrations of iron in the nutrient solution (e.g. browntop and sorghum). The iron treatments had no significant effect on the chlorophyll a to b ratio in the leaves. Iron concentrations. The concentration of iron in the leaves of all species increased with increasing concentrations of iron in the nutrient solution (Figs 3,4).

Iron requirements of C^ and C^ plants

547

I
"024 8 16 32 " 0.24 8 16 32 Iron concentrotion in nutrient solution {\ = O-'5\2 u.q Femr')

Fig. ]. Effect of iron in the nutrient solution on total dry matter yields (shoots plus roots) of Cg plants when nitrogen was provided either as ammonium nitrate (O) or as nitrate ( x }, Vertical bars indicate the residual standard deviation about the lines fitted to the observations.

0 124 8 ts 32 0 !Z4 8 16 32 Iron concentrotion in nutrient solution [liiO'3l2//g Femr')

Fig. 2. Effect of iron in the nutrient solution on total dry matter yields (shoots plus roots) of C^ plants when nitrogen was provided either as atnmonium nitrate (O) or as nitrate ( x ). Vertical bars indicate the residual standard deviations about the lines fitted to the observations.

However, greater concentrations of iron occurred in the leaves of plants grown on the ammonium nitrate-based solution than in leaves of those supplied with nitrate only as a source of nitrogen. The Cg plants had significantly greater concentrations of iron in their leaves than the C^ species, particularly when the concentration of iron in the nutrient solutions was highest. The results for the root (Figs 5,6) were broadly similar to those for the leaves except that the concentrations

548

G. S. SMITH et al.

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Iron requirements of Cg and C^ plants

(60 Browntop 14^! IZ8 (12 96 80 Lucerne

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024

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'"024

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Iron concentrotion in nutrient solution ((O-3(2^g Fe mf")

Fig. 3. Effect of iron in the nutrient solution on iron concentrations in the leaves of Cj, plants when nitrogen was provided either as ammonium nitrate (O) or as nitrate (x ). Arrows indicate iron concentrations associated with 90% maximum total dry matter yield. Vertical bars indicate the residual standard deviation about the lines fitted to the observations.

53

20
05 90 75 60 45 1 [

So'rghum

8 -^
1 1 1

i
.1

0'24 8 16 32 024 8 16 32 Iron concentration in nutrient solution (l = O-3l2/ig Fe m r ' )

Fig. 4 Effect of iron in the nutrient solution on iron concentrations ou. the leaves of C^ plants when nitrogen was provided either as arsimonium nitrate (O) oi" 3^s. nitrate ( x ) . Arrows indicate iron concentrations associated with 90% maxiinum total dry matter yield. Vertical bars indicate the residual standard deviation about the lines fitted to the observations.

were very much higher. The 'active iron' concentrations (i.e. that which was soluble in ether saturated 0-1 M HCl) averaged 15 to 20% of the total iron concentrations and followed similar patterns to those of the total iron concentrations in both shoots and roots (data not given). The concentration of iron in the leaves and in the nutrient solution, when the

55O
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2 4 8 i6 32 I E 4 8 1 6 32 Iron concentrotion in outrient solution (! = O-3l2^g Femf') (log scale)

Fig. 5. Effect of iron in the nutrient solution on iron concentrations in the roots of Cg plants when nitrogen was provided either as ammonium nitrate (O) or as nitrate (x). Vertical bars indicate the residual standard deviation about the lines fitted to the observations. The results were log transformed.

dry matter yield was 90 % of the maximum, was estimated for each species using the data in Figs 1 to 4. The results (Table 5) indicate little difference in the concentration of iron in the leaves among the species, but the C^ plants required more iron in the nutrient solution than the C^ plants to achieve these leaf concentrations. Phosphorus concentrations. Increasing concentrations of iron in the nutrient solution reduced the concentration of phosphorus in leaves, but not in the roots, of browntop, cocksfoot, ryegrass, kikuyu, maize and sorghum when grown on the ammonium nitrate-based solution (Table 6). Dry matter yields of all of these species were reduced by the higher concentrations of iron in the nutrient solution (Figs 1,2). Increasing the supply of iron greatly reduced the phosphorus: iron ratio in the leaves from relatively high values in the iron-deficient plants to values of less than 25 in plants whose dry matter yields were affected by excess iron (Table 6). Effects on other essential elements. Increasing concentrations of iron in the nutrient solutions had little or no effect on the concentrations of nitrogen, sulphur, potassium, magnesium, calcium, sodium or naanganese, in the leaves, apart from slight increases (10 to 15 %) in those plants affected by iron deficiency or excess

Iron requirements of C^ and C^ plants

800

551

3200 (600 800

600

j
J
I
t r

1 400 600300 f50-

300'
150 75'

^n

f 2 4 S [6 32 I .2 4 8 16 32 Iron concentration m nutnent solution [i = O'3!2^g Feml"-') (log scale)

Fig. 6. Effect of iron in the nutrient solution on iron concentrations in the roots of C^ plants when nitrogen was provided either as ammonium nitrate (O) or as nitrate ( x ).. Vertical bars indicate the residuaJ standard deviation about the lines fitted to the observations. The results were Jog transformeid..

(Table 7). Except for nitrogen, the concentrations of the elements were broadly similar for both the Cj and C, plants. For nitrogen, the concentrations were generally lower in the leaves of the C^ plants than in the leaves of the Cg plants. Manganese concentrations were slightly greater, while calcium concentrations were less, in the leaves of plants grown on the ammonium nitrate solution. The higher concentrations of calcium in the leaves of plants grown on the nitrate-only based solution reflect the greater quantities of calcium used in this nutrient solution (Table 2).
DISCUSSION

The concentrations of iron required in the nutrient solution for oiaximum growth of C^ plants were very much greater than those for Cg plants. Romheld & Marschner (1979) previously found that maize {Zea mays L. cv. Velox), a C^ plant, required concentrations of iron (as Fe EDDHA in the nutrient solution) up to 50 times greater than those required by sunflower (Helianthus annuus L. cv. Sobrid), a Cg plant, for maximum growth. However, no systematic study had previously been made of Cg and C^ plants. In the present investigation there were only small differences among the species in the concentration of iron required io the leaves for near maxinaum dry nriatter yield (Table 5). This suggests that the C3 and C^ plants differed in their capacity to absorb iron from the root zone, especially when nitrate was the only nitrogen source. The major form of iron potentially available to plants in well aerated soils is the ferric form (Fe^""") (Lindsay, 1972). However, it is the reduced ferrous form (Fe^^) which is absorbed by roots (Brown, Homes & Tiffin, 1961; Chaney, Brown & Tiffin, 1972). Attempts to classify plants into 'iron-efficient' or "iron-inefficient'

552

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Table 5. Concentration of iron required in the leaves and in the nutrient solution to achieve 90 % maximum dry matter yield of Cj and C^ plants grown either with ammonium nitrate or with nitrate only as the nitrogen source
Leaf iron (/ig Fe g ^ dry matter} Plant species C3 species Browntop Cocksfoot Perennial ryegrass Lucerne Red clover White clover C^ species Kikuyu Maize Paspaium Sorghum Mean C3 Mean C^ L S D {5 %) Ammonium nitrate Nitrate only Nutrient solution iron (/ig Fe ml"') .Ammonium nitrate Nitrate only

104 60
65 76 48

79 62
64 89 50 72 66

100 65 85 87 58 93
67 80

0-48 0-24 0-28 0-52 0-48 0-48 0-52 0-64 0-88 1-04 0-41 077 026

1-44 1-00 1-36 1-84 0>S6 1 60 2-32 5-96 7-04 4-08 1-30 4-85 2-0

83 62 81 73 21

27

types have been based on their capacity to reduce Fe^"*" to Fe^''" (Brown, 1976). In response to a deficiency of iron, ' iron-eflBcient' plants have a greater capacity to reduce iron fro^m. Fe^"*" to Fe^^ by lowering the pH at the root surface. This is probably caused by the release of H ions, organic acids such as citric acid, and reducing compounds such as riboflavin (Brown, Weber & Caldwell, 1967; Brown & Ambler, 1974; Romheld, Marschner & Kramer, 1982). Other factors, such as the form of nitrogen absorbed by plants, also influence the pH ofthe root zone. Numerous studies, including the present investigation, have shown that when ammonium is absorbed by plants there is an increase in acidity in the root zone as H ions exchange from the roots into the surrounding medium to compensate for excess cation uptake, whereas, with nitrate, large quantities of HCOg or OH ions are excreted by the roots to compensate for excess anion uptake (Trelease & Trelease, 1935; Dijkshoorn, 1962; Kirkby, 1969; Raven & Smith, 1976; Middleton & Smith, 1979). It seems likely that the higher iron requirements of both the C^ and Cj plants when nitrate was the source of nitrogen were due to less Fe^^ being formed as a result of the increased alkalinity of the root zone. Similar results have been noted with other plant species when they were grown on a nitrate-rich medium (Sideris & Young, 1946; Van Den Driessche, 1978; Foy, Fleming & Schwartz, 1981). However, in terms of 'efficiency' of the individual species,, there was no indication in the present experiment that the Cg plants were able to acidify the root zone to a greater extent than the C^ plants (Table 3). The possibility cannot be completely excluded, however, because it was not possible to sample the solution immediately adjacent to the root. There is also considerable evidence to suggest that high concentrations of phosphorus, calcium and naanganese interfere with the absorption of iron by the roots and with its subsequent transport from roots to shoots (Hewitt, 1963; Olsen, 1972). In this experiment it is unlikely

Iron requirements of Cj and C^ plants

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that these elements were present in high enough concentrations to have seriously affected the iron requirements of the Cj and C^ plants. Except for lower phosphorus concentrations in particular cases, the concentrations of all essential elements in the leaves were within the ranges considered to be optimal for growth of these types of plant (Benton-Jones, 1967; McNaught, 1970). The observed depressions in dry matter yield due to excess iron when ammonium nitrate was the nitrogen source were probably caused in part by a decrease in phosphorus uptake. The phosphorus concentrations in the leaves of plants severely affected by iron were below the optimal ranges for growth (Benton-Jones, 1967; McNaught, 1970). Similar effects of iron on phosphorus concentrations have been shown hy Dahiya & Singh (1976) and Brown & Jones (1977) for sorghum, and by Watanahe, Lindsay & Olsen (1965) for maize. Iron readily forms precipitates with phosphates and these may occur in the nutrient medium and on the surface ofthe roots (Sideris & Young, 1956), as well as internally (Rediske & Biddulph, 1953) thereby making both elements less available for plant growth. In the present study, iron appears to have affected the absorption of phosphorus by the roots to a far greater extent than transport from roots to shoots. If there had been a greater effect on transport to the leaves, a differential accumulation of phosphorus would have been expected to occur in the roots. In fact, increasing concentrations or iron in the nutrient solution had no significant effect on the concentrations of phosphorus in the root. There were also no distinctive leaf symptoms associated with the reductions in dry matter yields, although the development of brown lesions have been observed on the older leaves of rice (Tanaka, Loe & Navasero, 1966) and maize (Odurukwe & Maynard, 1969) affected by excess iron. Bienfait & Van Der Mark (1983) concluded that the regulation of Fe^""" in plants may also be of considerable importance in relation to the detrimental effects of iron on growth. Ferrous iron is readily oxidized by oxygen at pH values commonly found in plant ceils (pH 5-0 and ahove).

The superoxide (O^") that is formed in this reaction is highly reactive and can cause considerable damage to membranes and proteins, especially when there is insufficient superoxide dismutase present to keep the concentration of Ov," in the cells at low levels (Halliwell, 1974; Trelstad, Lawley & Holmes, 1981). The control mechanisms for maintaining low concentrations of Fe*"'' in the tissues may be less well developed in those plants whose growth was reduced by the relatively high concentrations of iron in the nutrient solution. Before a satisfactory explanation can be offered for the difference in capacity of the Cg and C^ plants to absorb iron, more detailed measurements are needed on the uptake of iron by roots of these two groups of plants. Furthermore, the study needs to be extended to include a much greater number of C3 and C^ plants.

ACKNOWLEDGEMENTS

We thank Carolyn Johnston and Carolyn Karl for technical assistance; Mr M. P. Upsdall for help with the statistical analyses; and the staff of the Plant and Analytical Chemistry Group for chemical analyses of the plant samples.

Iron requirements of Cg and C^ plants

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555

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