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Journal of Plant Nutrition

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Ferrous iron determination in

plant tissue
a b
E. E. Pierson & R. B. Clark
Department of Agronomy , University of
Nebraska , Lincoln, NE, 68583
U.S. Department of Agriculture , University of
Nebraska , Lincoln, NE, 68583
Published online: 21 Nov 2008.

To cite this article: E. E. Pierson & R. B. Clark (1984) Ferrous iron

determination in plant tissue, Journal of Plant Nutrition, 7:1-5, 107-116, DOI:

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JOURNAL OF PLANT NUTRITION, 7(1-5), 107-116 (1984)


Key Words: Iron nutrition, Sorghum bicolor (L.) Moench, Iron

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chelating agents, Leaf extractions, Mineral nutrition.

E. E. Pierson and R. B. Clark

Department of Agronomy
U.S. Department of Agriculture
University of Nebraska
Lincoln, NE 68583


Experiments were conducted to determine ferrous iron (iron 2 + ,

Fe ) in plant tissues. Iron2+ could be readily extracted from
leaf tissue with aqueous solutions of the chelating agents OPh
(1,10-o-phenanthroline) and PDTS [ferrozine; 3-(2-pyridyl)-5,6-bis-
(4-phenylsulfonic acid)-l,2,4-triazine]. Iron 2 values did not in-
crease when leaf tissues were extracted more than 30 minutes. The
first two extractions of the same tissue yielded higher Fe2+ values
than subsequent extractions. After the third extraction of the
same tissue, Fe2+ remained relatively constant. Even after seven
extractions of the same tissue, some Fe2+ could be extracted which
indicated that some iron (Fe) was probably being converted to Fe2+
with each extraction.

Initial extractions of the plant material had higher Fe2+ val-

ues when extracted with OPh than for PDTS, but subsequent extrac-
tions showed similar Fe2+ values for both extractants. Higher Fe2+
values were obtained from fresh leaf tissue than from freeze-dried

Published as Paper No. 7241, Journal Series, Nebraska Agricultural

Experiment Station, Project No. 12-095.


Copyright © 1984 by Marcel Dekker, Inc. O190-4167/84/0705-O1O7S3.5O/0


or oven-dried tissue; freeze-dried and oven-dried tissues gave

similar Fe2+ values. Total Fe measured in these tissues followed:
fresh > freeze-dried > oven-dried. After the second extraction of
the same tissue sample, Fe2+ extracted from each type of tissue
preparation were similar.


Early research indicated that chlorotic leaves contain as much

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as or more Fe than green leaves (Bennett, 1945; Oserkowsky, 1933).

This has been substantiated in many reports since that time (Hamze
and Nimah, 1982; Wallace et al., 1982). Because of this, it has
been suggested that "active Fe" and not total Fe is the important
fraction in plant tissues. "Active Fe" in plants has been reported
to be F e 2 + (Brown et al., 1979a; Clarkson and Hanson, 1980; Dekock,
1981; Katyal and Sharma, 1980; Olsen et al., 1982; Seckback, 1982),
and has also been defined in terms of the P/Fe ratio (Dekock et
al., 1979). "Active Fe" is considered to be that portion of Fe
available for, or participating in, metabolic reactions or incor-
porated into molecular structures. The remainder of the Fe is
probably precipitated and unavailable for plant use.

Katyal and Sharma (1980) reported a procedure for the determi-

nation of Fe * in plant tissues using OPh (1,10-o-phenanthroline)
to extract Fe . Significant differences between green and
chlorotic leaf tissue were noted; chlorotic leaves had less Fe
than green leaves. However, relationships between Fe and the ac-
tivity of Fe-enzymes or reactions have not yet been established.

Fresh plant leaf tissue and a 16-hour extraction time were

used in the the Katyal and Sharma (1980) method for F e 2 . The use
of fresh tissue would restrict Fe determinations to presently
growing plants; an important limitation for many investigations.
The 16-hour extraction time appeared to be long when reactions that
can occur with Fe are considered. For example, light can readily
convert F e 3 + to F e 2 + (Bennett et al., 1982; Brown et al., 1979a,
1979b; Krizek et al., 1982). The 16-hour extraction time also pro-
longs the analysis time. Another concern about the use of OPh as
the extractant is its nonspecificity for Fe . OPh is a well es-
tablished chelating agent used for the determination of total Fe in
soil extracts and plant tissues (Olson, 1965; McBride, 1980), and
it reacts extensively with Fe (Pierson and Clark, 1984).

The objectives of this study were: (1) to determine the time

required to quantitatively extract Fe from homogenized or ground
tissue; (2) to evaluate the extraction requirements; and (3) to
assess the applicability of the procedure for determining Fe in
both fresh and dried tissues.


1. Growth of plants. Sorghum [Sorghum blcolor (L.) Moench]

plants were used in this study. The plants were grown in nutrient
solutions in a growth chamber at 12-hour/I2-hour (light/dark)
cycles at temperatures of 28/23 ± 1°C. High pressure sodium
(General Electric, Lucalux LU 40088 1 ) and metal halide (General
Electric, Multivapor, MV400 1 ) lamps provided 450 yE m~ 2 sec" 1 PPFD
(photosynthetic photon flux density) at plant height (110 cm below
the light source). Humidity was maintained near 50% to assure ade-
quate plant transpiration in order to eliminate Ca deficiency symp-
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toms in the plants. Calcium deficiency is common in sorghum grown

in growth chambers and greenhouses under high humidities.

The nutrient solutions and techniques used for growing the

plant have been previously described (Clark, 1982). The composi-
tion of full-strength nutrient solutions was 22.9 NO3-N, 7.54 Ca,
7.24 K, 2.78 NH4-N, 1.55 Mg, 1.94 Cl, and 1.82 S in mmol/liter,
and 65 P, 49 Fe, 50 B, 18 Mn, 4.6 Zn, 1.6 Mo, and 1.2 Cu in
umol/liter. Iron was added as FeHEDTA [ferric N-2-(hydroxyethyl)-
ethylene-diaminetriacetate]. Five-day-old sorghum seedlings grown
from seeds germinated in wet paper towels were transferred to plas-
tic plates holding 120 plants and grown for an additional 14 days
in 0.5-strength nutrient solution. Nineteen-day-old plants were
transferred to plastic plates holding 10 plants and grown in full-
strength nutrient solutions with or without Fe (treatment solu-
tions). Nutrient solutions without Fe contained nitrate as the
lone source of N to cause the solution pH to rise and enhance the
induction of chlorotic leaves. Plants were grown in these treat-
ment solutions seven days.

2. Collection and processing of leaf samples. Terminal

leaves (emerging from the whorl) were collected from green and
chlorotic plants for the Fe determinations. Fresh samples were
processed immediately. For freeze-dried samples, fresh samples
were placed in paper bags, and dried at -45°C under vacuum for 48
hours. For oven-dried samples, fresh samples were placed in paper
bags and dried in a forced-air oven at 85°C for 48 hours. Freeze-
dried and oven-dried samples were placed in plastic vials and mas-
cerated using a fast-action reciprocal shaker. Teflon pellets were
enclosed in each vial for masceration. Maize (Zea mays L.) plants
were also used in experiments involving fresh, freeze-dried, and

1 Mention of trademark, proprietary product, or vendor does not

constitute a guarantee or warranty of the product by the University
of Nebraska and the U.S. Department of Agriculture, and does not
imply its approval to the exclusion of other products or venders
that may also be suitable.

oven-dried tissues. Maize plants were grown in a similar manner to

the sorghum plants.

3. Total iron determinations. Total Fe was determined on

wet-digested leaf samples of 400 mg fresh or 100 mg freeze-dried or
oven-dried tissue. Samples were digested with concentrated H 2 S 0 A
(2.0 ml) and 30% H 2 0 2 (2.0 ml) in 22 mm x 25 cm test tubes at 350°C
on a heating block. Additional H 2 0 2 was added to digest organic
matter until solutions were clear. The digest solutions were cool-
ed to ambient temperature and reagents added in the following se-
quence to each tube: 2.0 nl of 1.4 M hydroxylamine-HCl, 2.0 ml of
0.076 H OPh in 95% ethanol, and 8.0 ml of 4.0 M NH4OH (pH 2.5 to
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3.5) (Olson, 1965; Saywell and. Cunningham, 1937). Other reagent

addition sequences decreased the color intensity. This was because
hydroxylamine must be added first to reduce any Fe to Fe before
reaction with OPh (a Fe chelating agent). The contents of each
tube were transferred to 25.0 ml volumetric flasks. The solutions
were brought to volume, shaken, and spectrophotometric absorbance
readings obtained at 510 nm. Four replicate samples from each
treatment were analyzed.

4. Leaf extractions for Fe . Tests to determine the time

and the number of extractions required to extract Fe from the
leaf samples were performed on oven-dried sorghum tissue. To 100
mg mascerated tissue in 50 ml Erlenmeyer flasks was added 2.0 ml of
the appropriate Fe^ + chelating agent and 15 ml distilled water.
The Fe^ + chelating agents were OPh and PDTS [ferrozine; 3-
(2-pyridyl)-5, 6-bis-(4-phenylsulfonic acid)-l,2,4-triazine].
Flasks were shaken for different lengths of time on a slow action
reciprocal shaker. Their contents were filtered under a slight
vacuum through Whatman No. 1 filter paper into 25.0 ml volumetric
flasks. Each filter paper with its respective insoluble leaf
sample residue was put back into its Erlenmeyer flask and re-
extracted as described above. Seven extractions were made on each
sample. Four replications of each experiment were performed.

After the time required for extraction was evaluated, fresh,

freeze-dried, and oven-dried leaf tissue were analyzed for Fe .
Fresh leaf tissue (400 mg) was homogenized in 40 ml glass tissue
grinders (Ten Brock ) with 2.0 ml of chelating agent. The homo-
genate and subsequent rinses were transferred to 50 ml Erlenmeyer
flasks. One hundred mg samples of mascerated freeze-dried or
over-dried leaf tissue were weighed into 50 ml Erlenmeyer flasks
and 2.0 ml of chelating agent and 15 ml distilled water were added.
The Erlenmeyer flasks containing the fresh, freeze-dried, and oven-
dried tissues were shaken on a slow-action reciprocal shaker for 30
min. Contents of each flask were filtered into volumetric flasks
as described above. The tissues were extracted.three times. Four
replications of each type of tissue preparation or treatment were

The chelating agents used for Fe^ extractions were prepared

as follows: 15 g of OPh was added to 850 ml distilled water, 1 _N

HC1 was added dropwise until the salt dissolved (pH 3.5 to 5.5),
and the volume brought to 1 liter (0.076 M ) . PDTS (5.14 g) was
dissolved in 100 ml water, 500 ml of concentrated HC1 was added,
the solution was cooled, and brought to 1 liter (0.10 tl).

5. Determination of F e 2 + . Filtered solutions of OPh extracted

leaf tissue were brought to 25.0 ml volume with distilled water and
the absorbances were read at 510 nm (Katyal and Sharma, 1980).
Filtered solutions of PDTS extracted leaf tissue were heated for 10
min in a 98°C water bath, cooled to ambient temperature, and 2.0 ml
of 5.2 H ammonium acetate buffer was added to each sample (McBride,
1980). Solutions were brought to 25.0 ml volume with distilled
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water and absorbances read at 562 nm. Additional procedure and re-
agent preparation details are discussed by Pierson and Clark


No differences in Fe values were observed when oven-dried
sorghum leaf samples were extracted for 0.25, 0.50, 1.0, or 2.0
hours with PDTS or OPh (Table 1 ) , Leaf tissues extracted for 0.5,

Table 1. Effects of extraction time on Fe determinations using

PDTS and OPh extracts of oven-dried sorghum leaf tissue.

Extraction Extraction agent


Hours Vig Fe 2+ /g dry wt.

Experiment I

0.25 9.8 a* 10.9 a

0.50 11.2 a 11.6 a
1.0 10.5 a 11.7 a
2.0 11.8 a 11.5 a

Experiment II

0.5 17.3 a 11.4 a

4.0 19.2 b 13.6 b
8.0 18.2 ab 14.2 b
16.0 18.5 ab 14.7 b
24.0 20.5 c 14.5 b

^Numbers followed by different letters are significant at P = 0.05

according to Duncan's multiple range test.

4, 8, 16 and 24 hours (Table 1) showed slightly lower F e 2 + values

for the 0.5-hour extraction time than for longer extraction times,
but these higher values were not consistently significant. The Fe
values for the different extraction times were within 20% of each
other for PDTS extracted tissue and within 30% for the OPh
extracted tissue. The slightly higher F e ^ + values measured for
tissue that were extracted for longer times may have been due to
some tissue Fe conversion to Fe during the extraction process.
Subsequent studies indicated that some Fe could be extracted from
leaf tissue after several extractions of the same tissue (Table 2 ) .
From these data, it was concluded that tissues need to be extracted
for only a short time (0.5-hour) to extract Fe from leaves, and
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that longer extraction times were of no particular advantage.

Reextractlon experiments with PDTS or OVh showed the first ex-

traction to have two to five times higher Fe values than in the
second extraction (Table 2 ) . Subsequent Fe extractions by OPh

Table 2. lionr+ values for subsequent extractions of the same green

and chlorotic freeze-drled sorghum leaf tissues extracted
with PDTS and OPh.

Extraction Green leaf tissue Chlorotic leaf tissue

number PDTS OPh PDTS OPh

yg Fe 2 + /g dry wt.

First 12.3 a* 20.6 a 7.7 a 12.6 a

Second 7.2 b 4.6 b 3.0 b 2.5 b
Third 3.2 c 4.1 be 2.2 b 2.6 b
Fourth 3.1 c 3.0 dc 2.2 b 1.1 b
Fifth 2.7 dc 2.9 cd 1.8 b 2.8 b
Sixth 2.8 c 3.4 cd 3.1 b 2.2 b
Seventh 2.1 d 2.8 d 1.8 b 2.3 b

CV (%) 26 53 45 64
Total F e 2 + 33.3 B* 41.4 A 22.8 D 26.0 C
(by addition)

Total Fe 124.2 26.5

(from digests)

'Numbers followed by different small letters in each column are

significant at P = 0.05 according to Duncan's multiple range

^Numbers followed by different capital letters are significant at P

= 0.05 according to Duncan's multiple range test.

and PDTS remained relatively constant. The F e 2 + values from the

first extraction were higher for OPh-extracted tissue than for
PDTS-extracted tissue. The various extractions were summed to yield
total Fe 2 (Table 2 ) . The differences between PDTS and QPh extract-
ed samples could be attributed to the differences in Fe values
for the first extraction. Reasons for the higher Fe values for
OPh extracted tissue are unknown, but studies with standard solu-
tions (Pierson and Clark, 1984) showed that OPh could react with
more Fe than PDTS. Whether these differences were due to OPh
reaction with Fe is uncertain.

Even after seven extractions of the same tissue sample, some

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Fe was present in the extracting solution regardless of chelating

agent used (Table 2 ) . Iron values after the second extraction
were similar. These data indicated that with subsequent extraction
of the same tissue either some of the Fe (probably Fe ) within the
tissue was converted to Fe or that all of the Fe was not being
extracted in the initial extractions. The former explanation ap-
pears to be more feasible since Fe values remained relatively
constant with each subsequent extraction. At no time did the Fe 2
values become nil. Additional studies showed that Fe values of
subsequently extracted tissue were the same whether they were ex-
tracted immediately following a previous extraction or whether some
time (24 to 72 hours) elapsed before other extractions occurred
(data not reported).

Extraction of fresh, freeze-dried, and oven-dried sorghum and

maize leaves showed similar results to those mentioned above (data
not reported). Tissues extracted sequentially several times con-
tinued to yield some F e 2 + whether extracted with PDTS or OPh.

The coefficient of variation (CV) and standard deviations

among the Fe values were lower for PDTS than for OPh extracted
tissue (Table 2 ) .
Fresh sorghum and maize leaf tissues yielded higher Fe and
total Fe values than freeze-dried or oven-dried tissues (Tables 3
and 4 ) . Freeze-dried and oven-dried green sorghum (Table 3) leaf
tissue and freeze-dried and oven-dried green and chlorotic maize
(Table 4) leaf tissue gave similar F e ^ + values, but freeze-dried
green maize leaf tissue had higher Fe values than oven-dried tis-
sue (Table 4 ) . Total Fe values for both sorghum and maize were
different and consistent for each tissue preparation: fresh >
freeze-dried > oven-dried. The differences in total Fe among tis-
sue preparations were not expected, but with each tissue prepara-
tion, the results were similar. Reasons for the higher total Fe
extracted from fresh tissue than in freeze-dried or oven-dried tis-
sue are not known.
PDTS and OPh were selected as leaf tissue extractants for Fe
in these studies because of their ease in assay procedures, detec-
tion of Fe over wide ranges of Fe concentrations, and lack of

Table 3. Iron and total Fe values for fresh, freeze-dried, and

oven-dried green sorghum leaf tissue.
OPh Fe z Total Fe

Ug Fe/g dry wt.

Fresh* 29 .8 a§ 27.4 a 153 .0 a

Freeze-dried 16.6 b 12.2 b 91 .8 b
Oven-dried 16.0 b 11.1 b 72 .6 c
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'Samples for each preparation were from the same bulk leaf sample.

*Dry weights of fresh samples were calculated by determining

moisture percentages in separate portions of the same bulk leaf
sample (by weight).

^Numbers followed by a different letter in each column are

significant at P = 0.05 according to Duncan's multiple range test.

Table 4. Iron and total Fe values for fresh, freeze-dried and

oven-dried green and chlorotic maize leaf tissue.

Tissuet Green tissue Chlorotic tissue

preparation PDTS F e 2 + Total Fe PDTS Fe 2 h Total Fe

5 Fe/g dry wt •
Fresh* 30 .6 a 177 a 36 .0 a 60.2 a
Freeze-dried 20.4 b 154 b 12.6 b 45.3 b
Oven-dried 17.9 c 123 c 12.7 b 32.4 c

t,*,§ Same as for Table 3.

interferences by P and other elements/compounds commonly found in

plant tissues (Pierson and Clark, 1984). In these studies, the
experimental results for both PDTS and OPh were similar except that
PDTS yielded lower CV values (Table 2 ) . In addition, PDTS did not
react with Fe as readily and gave higher absorbance readings for
the same amount of Fe as OPh. PDTS was considered to be the pre-
ferred Fe extraction agent for Fe determinations in leaf tis-

Our objectives were to determine extraction procedures for

Fe , to evaluate extraction times of plant samples, and to deter-

mine whether freeze-dried or oven-dried tissues could be used in-

2+ 2+
stead of fresh tissue for Fe determinations. Iron could be ex-
tracted from leaf tissues, but repeated extraction of the same tis-
sue gave additional amounts of Fe . Since Fe values remained
constant after the second extraction of the same tissue, three ex-
tractions were considered adequate to obtain consistent Fe
values. An extraction time of 0.5-hour was as effective as 16-
hours [the time used by Katyal and Sharma (1980)]. Thus, the ex-
traction time for Fe determinations of plant tissues could be
shortened extensively. Even though fresh tissue yielded higher
Fe values than freeze-dried or oven-dried tissues, dried tissues
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could be used without difficulty for Fe determinations.

In summary, Fe determinations of leaf tissues can be ob-
tained with relative ease. If the number of Fe extractions are
defined, Fe values should be meaningful for plant tissues.


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