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Variation in Localization and Accumulation of
Iron in Pigeon Pea Seeds under the Influence of
Different Strategies of Iron Application
By
Rajni Shukla, Saumya Srivastava, Yogesh Kumar Sharma
and Arvind Kumar Shukla
ISSN 0970-4973 Print
ISSN 2319-3077 Online/Electronic
Global Impact factor of Journal: 0.756
Scientific Journals Impact Factor: 2.597
Index Copernicus International Value
IC Value of Journal 4.21 Poland, Europe
J. Biol. Chem. Research
Volume 32 (1) 2015 Pages No. 100-112
Journal of
Biological and
Chemical Research
An International Journal of Life Sciences and Chemistry
Indexed, Abstracted and Cited in Various National and International Scientific
Databases of the World
Published by Society for Advancement of Sciences®
J. Biol. Chem. Research. Vol. 32, No. 1: 100-112, 2015
(An International Journal of Life Sciences and Chemistry)
Ms 32/1/25/2015, All rights reserved
ISSN 0970-4973 (Print)
ISSN 2319-3077 (Online/Electronic)
Prof. Y. K. Sharma
http:// www.jbcr.in
jbiolchemres@gmail.com
info@jbcr.in
Received: 01/12/2014
Revised: 21/12/2014
RESEARCH PAPER
Accepted: 22/12/2014
Variation in Localization and Accumulation of Iron in
Pigeon Pea Seeds under the Influence of Different
Strategies of Iron Application
Rajni Shukla, Saumya Srivastava, Yogesh Kumar Sharma
and *Arvind Kumar Shukla
ICAR-NAIP, Department of Botany, Lucknow University, Lucknow- 226 007, U.P., India
*Indian Institute of soil science Bhopal, M.P., India
ABSTRACT
The availability of iron ultimately affects human both in terms of crop yield and the iron
concentration of edible tissues. As the plant based diet offers relatively low amounts of
bio-available iron (Fe), its deficiency is a serious nutritional problem in an estimated 30%
of the world population. Particularly our aim was to screen the different pigeon pea
varieties, commonly grown in subtropical climatic conditions of India, regarding high iron
accumulation and localization in grains. In Pigeon pea, among the four varieties BDN-2,
PKV-TROMBAY, C-11 and AAUT-2007-8 chosen for the investigation purpose, based on
their yield performance during previous years, the variety AAUT-2007-8 was found to
contain highest Fe contents in seeds. The findings were based on seed samples collected
from field experiments conducted in Anand Agriculture University, Gujarat, which were
subjected to FAA fixing, alcoholic dehydration, wax embedding, microtomy and staining
with Prussian blue followed by image analysis in transmitted light microscopy. The field
treatments included the control (normal soil), iron application in soil and iron application
in soil with foliar spray of iron on three different stages namely at pre-flowering,
flowering and grain filling stages. The quantitative analysis of iron in the seed parts
clearly exhibited that most of the iron in PKV-TROMBAY was confined to the seed coat
that is 217.50 as compared to 118.94 (observed in terms of color intensity of the stain) in
cotyledons. For the point of view of high iron localization in cotyledons the variety AAUT2007-8 was found best among all the four varieties undertaken in the study, as it showed
maximum iron in cotyledons that is 199.49. Among the field treatments the foliar
applications were given in addition to soil application of iron which was found most
effective regarding iron accumulation in seeds. The technique proved to be a cost effective
and time saving for the screening of varieties for nutrient accumulation as well as their
distribution in different plant parts as compared to any other methods like Atomic
Absorption Spectrophotometer etc.
Key words: Iron, Accumulation, Prussian Blue, Image Analysis and Alcohol Dehydration.
Variation……………………………………………………Application
Shukla et. al., 2015
INTRODUCTION
As a common practice and need for human and animals various plant species and their
different cultivars are grown for procuring their valuable produce to human welfare
specially the reserve food as carbohydrates, proteins, fats or some alkaloids. It may be for
the leaves containing alkaloids as tea, tobacco etc. or root/shoot providing high amount of
carbohydrates like beta roots and sugarcane etc. or the fruits or seeds for their high protein,
carbohydrate or fat contents, and other nutritious value as nutrients or vitamins. Due to
developing and cultivating high yield varieties the soils became nutrient deficient and it
became a serious concern to enhance the nutrient value of plants. Iron is one of the
essential nutrients for plants and human. The availability of iron ultimately affects human
both in terms of crop yield and the iron concentration of edible tissues. As the plant based
diet offers relatively low amounts of bio-available iron, its deficiency is a serious nutritional
problem in an estimated 30% of the world population. Thus a large portion of the
developing world suffers from Fe deficiency, including over 60% of all children in Africa and
South East Asia. Inadequate Fe in diet can cause serious health problems such as anemia,
impaired immune system and poor cognitive function. It is estimated that Fe deficiency
exists in about half of the world population (Welch and Graham, 2004). The crop breeding
has often selected the programs for disease resistance, high yield and improved seed
maturation time, but ignored nutrient accumulation in the seeds as a desirable trait.
Although the existence of a large and useful genetic variation is of great importance for a
successful breeding program aiming at improving seeds with micronutrient. Studies with
limited germplasm have shown large genetic variability for their indicated traits, providing
good opportunities to select genotype with still higher grain Fe. (Abdulla et al., 1998). Iron is
essential for plants. It is highly reactive and toxic via the Fenton reaction. The plants tightly
control Fe homeostasis and react to Fe deficiency as well as iron load. Plants mainly acquire
Fe from the rhizosphere. Although Fe is one of the most abundant metals in the earth's
crust, its availability to plant roots is very low. Although various mechanisms for iron intake
have been worked out but still its absorption and mobility in plant system is very low. Fe
availability is dictated by the soil redox potential and pH. In soils that are aerobic or of
higher pH, Fe is readily oxidized, and is predominately in the form of insoluble ferric oxides.
At lower pH, the ferric Fe is freed from the oxide, and its availability becomes more for
uptake by roots. Many food crops are agronomically productive, but have low levels of
nutrients like Fe in the seed. Increasing the concentration of micronutrients in food crops is
a growing global challenge with great amplification for both crop production and human
health. The leguminous plants are the best source of proteins and mineral elements for
human well being particularly in vegetarian diets. Pigeon pea is a very popular crop which
shows high consumption all over India. It provides high protein contents. Structurally Pigeon
pea seed consists of cotyledon 85%, embryo 1% and seed coat 14% (Faris and Singh, 1990)
of its total size or volume. Pigeon pea also contains some unavailable carbohydrates which
reduce the bioavailability of other nutrients. (Komoth and Belavady, 1980). Green pigeon
pea is a packed source of iron (Singh et al., 1984). Dehulling of pigeon pea removes about
20% of calcium and 30% iron (Singh and Jambunathan, 1990).
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Although supplements added to food or taken in tablet form are effective in preventing and
controlling iron deficiency but such treatments are difficult to implement in developing
countries because of the associated cost and the small number of the primary health care
programs. Some crops like spinach and legumes are better known for their high iron
content. However, these plants also contain oxalic acid and phytate like substances that
decrease their bioavailability. A reduction in the amounts of phytic acid, therefore, is
regarded as an important strategy for improving iron and zinc bioavailability (Ruel, and
Bouis, 1998). In this scenario enhancing grain iron (Fe) content is one of the effective ways
of increasing the Fe intake and reducing the Fe deficiency anemia in human (Welch and
Graham, 2002).
Among the existing and in-practice methods the plant samples are tested for iron contents
using Atomic Absorption Spectrophotometer (AAS) or Inductively Coupled Plasma Atomic
Absorbtion Spectrophotometer (ICP). The bottleneck in this process is the high cost of Fe
done with digestive method which requires relatively expensive instruments and very time
consuming. A procedure based on Perl’s Prussians blue stain was used for rapid screening
of iron content in pigeon pea as adopted in rice by (Krishnan et al., 2003) which involves
scoring color intensity in the embryo of cut and treated seeds (with 2% Prussion blue). The
objective of this research is based on the screening of growing pigeon pea variety of high Fe
content and after iron treatment through soil and foliar application, identifying the variety
performing better regarding Fe content. As per literature available the large amounts of
nutrients like zinc and iron remain localized in seed coat while only a small fraction is stored
in embryo. As a most light component of seed the seed coat is removed during soaking and
cooking pigeon pea resulting thereby only a very little iron availability in human food. In
response to this growing problem of iron deficiency in vegetarian diets, this research has
been undertaken to focus on screening of the high iron containing varieties, and
understanding the iron
accumulation and distribution in different seed parts. Using
variable Fe application strategies, an effort has been made to to achieve the goal which will
enhance the Fe localization and accumulation in cotyledons. The efforts are made in this
study to enhance the iron localization in embryo and cotyledons using different methods of
iron application in plants at different stages, hence ultimately reaching more iron in human
food as Pigeon pea is one of the commonest pulses in vegetarian diet.
MATERIAL AND METHODS
During the kharif season of 2009, a field experiment was conducted in Anand Agriculture
University, Anand (Gujarat) India for the screening of certain varieties for high iron
accumulation in Pigeon pea (Cajanus cajan) seeds in response to different strategies
adopted for iron application. Among the treatments, control (normal soil of the area- T1), 20
kg/ha FeSO4 soil application –(T2), and soil Fe application followed by 0.5% FeSO4 foliar
application–(T3) at three different growth stages were undertaken with three replicates in a
random/split plot/design/rows. All the standard agronomic practices were adopted for the
irrigation and other timely necessary measures in the field. The plant samples were
collected at different stages, and finally the mature seeds were harvested for the
experimental purpose.
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The collected seed samples were fixed and processed at Lucknow University centre for the
accumulation and localization studies of iron using microtomy, staining and image analysis.
The seeds were taken from four different varieties on the basis of their high and low
efficiency for past yield performance, and they were- BDN-2, C-11, AAUT-2007-8 and PKVTROMBAY. To use the Perls Prussian blue staining procedure for iron accumulation and
localization through anatomical technique, all the precautions were taken to avoid external
contamination of seeds with dust in the field during threshing and clearing. For the purpose
the staining method as modified by Ozturk et al. (2006); Lunal (1968); Sheehan and
Hrapchak (1960); Crookham and Dapson (1991) was used.
The reagents were prepared as follows
Aqueous potassium ferrocyanide (10%) (A)
50 g potassium ferrocyanide was mixed with distilled water and the volume was made to
500 ml. The solution was transferred into acid cleaned brown bottle for storage which
remains stable for six months.
Aqueous hydrochloric acid (10%) (B)
50 ml concentrated hydrocholoric acid (HCl) was mixed with distilled water to make the
volume 500 ml. This dilute HCl was transferred into brown bottle for storage which remains
stable six months.
Perl’s Prussian blue solution
The solution was prepared by mixing 10% potassium ferrocyanide (A) and 10% HCl (B) in 2:1
ratio, respectively. The solution was freshly prepared every time during use. (Ozturk et al.,
2006).
Cutting
Seeds were soaked for few hours and after that they were subjected to the alcoholic
dehydration series using ethyl alcohol. After that they were embedded in paraffin wax block
and sections were cut through microtome (Yorco-YSI-062) of 100 µM thickness.
Staining for Fe localization
Staining of seeds sections for Fe localization was performed at room temperature for 30
minute.
Image analysis
The stained seed sections were examined through Image analyzer and analysis was done by
NIS-ELEMENT, BR 3.1 software program, and photographs were taken by using a
transmitted light microscope with a high resolution digital camera (both Nikon, Japan).
RESULTS
The results obtained in the experiment are shown in table 1 and 2, figures 1 and 2, and
photoplates 1 and 2. Perls Prussian blue staining for Fe resulted in greenish blue color. The
Fe localization method applied in this study was not only helpful in locating the Fe-rich seed
part as well as it can be used as a practical rescreening method for selection of genotype.
The variety AAUT-2007-8 and PKV-TROMBAY were identified better in comparison to other
varieties like C-11 and BDN-2 in relation to the Fe localization and accumulation.
The data regarding iron localization in seeds (as shown in table 1) clearly indicate that
variety AAUT-2007-8 performed its best as the iron content (shown in comparative manner)
was maximum that is 416.56 (as color intensity of the stain) in control.
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The other varieties like BDN-2, PKV-TROMBAY and C-11, their values were 209.68, 336.44
and 329.72 respectively. These results show that the variety BDN-2 shows poorest
localization of iron. In terms of efficiency the BDN-2 shows least efficiency and AAUT-200708 show highest efficiency. If the total seed content is measured the highest enhancement
of localization of iron shows in terms of color intensity in BDN-2 that is 52.73 and 90.99 in
soil application (T2) and soil + foliar application (T3) respectively but PKV-TROMBAY shows
highest enhancement in accumulation of iron in cotyledon that is 45.77 in T2, 99.68 in T3
respectively and AAUT-2007-8 shows less enhancement of iron in soil application (T2) and
soil + foliar application (T3) as shown in figure 1.
Out of this total iron in seeds an important significant fraction of iron remains confined to
seed coat while remaining fraction is localized in cotyledon. As per the data shown in Table1 AAUT 2007-8 varities shows highest accumulation in control treatment as well as in soil
application and soil + foliar application regarding localization of iron. The varieties BDN-2
and C-11 shows less enrichment that is 12.66-T2 and 1.002-T3 and 33.02- T2, 51.20-T3
respectively in cotyledon. The above variety shows more efficiency in comparison to AAUT2007-8 and less efficiency from PKV-TROMBAY is (45.77-T2, 99.68-T3) in terms of Fe
localization. The BDN-2 variety shows maximum accumulation in seed coat through soil
application (104.66)-T2 and soil +foliar application (129.84)-T3 but it is unvalued because
seed coat is never used for eating purpose.
The iron contents in seeds of iron applied soil and iron applied soil with foliar iron
application clearly indicated the enhanced iron localization and accumulation in all the
varieties as compared to their respective controls (Table- 1 and figure -1). The iron
contents measured in seed digest using double AAS are shown in Table-2 which clearly
indicates the AAUT-2007-8 as a high iron accumulating variety even in control. The total iron
contents in seeds in this variety were reported 37.4 ppm while in BDN-2, PKV-TROMBAY and
C-11 the iron contents were 32.8, 32.7 and 34.8, respectively. The iron application in soil
enhanced the accumulation of iron in seeds in PKV-TROMBAY, C-11 and AAUT-2007-8 as it
was observed 34.8, 36.1 and 40.1 in respective three varieties although BDN-2 did not
respond to any iron application in soil with foliar iron application. These three varieties
showed further enhanced level of iron in seeds as the iron contents reported were 36.4,
39.4 and 40.9 respectively in three varieties. The BDN-2 did not show any positive increase
in iron contents instead it showed reduced level of iron in seeds which was in contradiction
to the results obtained through staining procedure. The results obtained in staining and
image analysis of the seeds and its different parts (as shown in Table-1 and Photoplates 1
and 2 clearly conclude the enhancement of the iron accumulation in seeds and its
localization in seed coat and cotyledon. In the observations based on staining , image
analysis and comparative degree of iron stain intensity (Figure-1) the variety PKV-TROMBAY
in T3 shows highest accumulation of iron in cotyledon (the edible part of the seed ) as it
was found to be increased by 99.68 % over its control (T1) , even iron in seed coat was
found to be reduced by 7.15% in T3 over its control T1. It gave a clear idea that it moved
more to cotyledons. Another variety AAUT-2007-8 also showed increase in iron localization
in cotyledon by 41.20% in T3 over its control (T1) but simultaneously it also showed
increased localization in seed coat by 6.5% in T3 over its control (T1).
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DISCUSSION
The results obtained in the experiment clearly show the iron contents accumulated in whole
seed and in different seed parts in reference to different varieties and treatments. This iron
absorption by the plant from the soil or through foliar application and its translocation
within the plant system follow a particular strategy which may be interpreted in different
ways.
Under reduction based strategy in non-gramenaceous plants (Eckahardt et al., 2001; Li et
al., 2004, Romheld and Marschner (1983) and Waters et al. ( 2007) during iron deficiency
the protons are released into the rhizosphere by root epidermis which lowers the soil pH
making iron more soluble. The NADPH dependent ferric chelate reductase then reduces
Fe+3 to Fe+2 (Robinson et al., 1999). This reduced Fe(II) can then be transported into the
root epidermal cells by the divalent metal transporter (Henriques et al., 2002,Vert et al.,
2002,Varotto et al., 2002) which also transports other metals like Zn, Mn, Cd, Co Eide et al.,
1996; Korshunova et al., 1999 and Ni (Schaaf et al., 2006) etc. The other aspect of Fe
deficiency response in non-gramenaceous plants is secretion of phenolic compounds into
rhizosphere (Marschner, 1995) and the uptake of apoplastic iron which is later mobilized
into the symplast (Zhang et al., 1991).
Chelation based strategy on Fe uptake in grasses depends on its chelation by soluble
siderophores exuded from root epidermis, which have high affinity for Fe3+ (2). The
resulting FeIII-PS complexes are readily transported into the root epidermis via a high
affinity uptake system (Curie et al., 2001). Although some other gramenaceous species like
rice combines components of both the reduction strategy with Fe-PS uptake (Inoue et al.,
2009, Lee et al., 2009). Nicotianamine (NA) levels have a significant positive effect on metal
homeostasis. If NA level is increased accumulation of iron is also increased (Takahashi,et.al.
2003). Conversely, the loss of NA leads to Fe deficiency symptoms like interveinal chlorosis,
reduced growth and sterility (Takahashi et al., 2003, Klatte et al., 2009, Ling et al., 1999). Fe
moves to the seeds via the phloem as the flow of xylem is driven by transpiration and seeds
do not transpire (Grusak, 1994)). The timings and regulation of senescence have a
significant effect on Fe accumulation in seeds. Fe-NA is essential for flower and seed
development. The loss or depletion of NA results in deformed flowers and sterility as well as
significant decrease in floral Fe accumulation.
In legumes 90% of iron is stored in ferritin (Briat et .al., 1999) in seeds. Although Fe bound
by phytate or some other form of chelatores in the vacuole is also reported (Ravet
et.al.2009). Disruption of phytate biosynthesis and increased uptake of Fe through foliar
application at a specific growth stage may enhance the bioavailability and accumulation or
over expression of ferritin in seeds.
This iron staining technology using Prussion blue stain has also been used in others crops
like wheat, pearl millet, rice (Ozturk et al., 2009; Shivprakash et al., 2006; Pramuthai et al.,
2006). This Perl’s Prussian staining has been recommended as a method for locating Fe (III)
in tissue to give a distinctive blue reaction product (Baker, 1958). The technique has recently
been used. The efforts for detecting the iron in these crops like Rice, Wheat, Pearlmillet
have been made by Krishnan et al., 2001, Chanakan et al., 2003; Ozturk et al., 2009; Velu
et al., 2006.
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Table 1. Fe localization in seeds and its different parts in Pigeon pea (Cajanus cajan).
Varieties
Of Pigeon
Pea
BDN-2
PKVTROMBAY
Control
Control +Soil Application
Control + Soil Application+ Foliar Spray
Seeds
Seed coat
Cotyledon
Seed
Seed coat
Cotyledon
Seeds
Seed coat
Cotyledon
209.68
336.44
91.34
217.50
118.34
118.94
320.26
396.39
186.94
223.28
133.32
173.11
400.47
439.44
209.94
201.94
190.53
237.50
C-11
329.72
209.94
119.78
375.69
216.36
159.33
410.33
226.84
181.11
AAUT2007-8
416.56
217.07
199.49
432.61
217.27
215.27
479.90
231.18
248.72
Table 2. Fe content (ppm) in seeds as measured by AAS (Atomic Absorbtion
Spectrophotometer) in different varieties at different stages of iron application.
Varieties
Control
BDN-2
PKV-TROMBAY
C-11
AAUT-2007-8
32.8
32.7
34.8
37.4
Treatments
Control
+
Soil Control + Soil Application +
Application
Foliar Spray
32.6
29.9
34.8
36.4
36.1
39.4
40.1
40.9
Figure 1. Percent increase or decrease in Fe content in seed coat, cotyledon and seeds.
Treatment
Control + Soil Application
Control + Soil Application +Foliar Spray
Varities
SEEDCOAT
COTYLEDON
SEED S
SEED
COAT
COTYLEDO
N
SEED S
BDN-2
+104.66
+12.66
+52.73
+129.84
+61.002
+90.99
PKVTROMBAY
C-11
+2.66
+45.77
+17.81
-7.15
+99.68
+30.61
+3.058
+33.02
+13.94
+9.1835
+51.20
+24.44
AAUT-2007- +0.124
08
+7.010
+3.85
+6.50
+41.20
+15.20
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The level of iron is not exactly the same for every variety but variations were observed
subject to the soil properties and mode of application. The results obtained in the present
study are in correlation with the results of Gregario et al. (2000) who observed the
increased iron level with increase in iron application. Increasing the iron concentration
through soil application and foliar spray no doubt enhances the iron contents in seeds which
lead to reduce the iron deficiency in human being. More over the Plant’s ability to
translocate and absorb iron is genetically controlled (Brown et al., 1988), which may be
easily selected through screening the large number of varieties and assaying the iron
contents and its localization in different seed parts using this Prussion staining method.
These two physiological and genetical factors inconjuction might present an unlimited
translocated of iron to the seeds and reduce the theoretical amount of iron that could be
accumulated. A nutritional approach whereby available dietary iron is held at an adequate
level is the ultimate solution to prevent iron deficiency. Welch, R.M. and Graham, R.D. 2004.
Breeding for micronutrients in staple food crops from a human.
Enhancing the iron content is a good strategy as an alternate to iron fortification, which is
commonly not available to the neediest population (Walter et.al., 1997). The seeds of
certain varieties of pigeon pea at mature stage showed the localization of maximum amount
of iron that is the seed coat part. The pigeon pea seeds section showed the localization of
maximum amount of iron that is seed coat and cotyledon part. The pigeon pea seeds section
showed a positive reaction to the Prussian blue test for ferric iron along the outer region of
scuttelum and aleurone. Cotyledons of the red gram also react with Prussian blue test
showing the most intense colour along the peripheral area. The result is correlated and
colour is similar with the result of Jacob and Walker (1977) in maize. The soils as well as
foliar application of iron were undoubtedly associated with the iron accumulation and
localization in cotyledons as observed by staining technique.
120
% increase in soil application
100
80
60
SEED COAT
COTYLEDON
40
SEED
20
0
BDN-2
J. Biol. Chem. Research
PKV-TROMBAY
107
C-11
AAUT-2007-8
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Shukla et. al., 2015
140
120
% increase through foliar application
100
80
SEED COAT
COTYLEDON
60
SEED
40
20
0
BDN-2
PKV-TROMBAY
C-11
AAUT-2007-8
-20
PLATE-1
BDN 2 T1
PKV Trombay T1
BDN 2 T2
PKV Trombay T2
BDN 2 T3
PKV Trombay T3
Fe accumutaion in seeds of pigeon pea varieties.
T1: Control; T2: 20 kg/ha FeSO4 soil application; T3: Soil Fe application along with 0.5%
FeSO4 foliar application.
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PLATE 2
C11 T1
AAUT 2007-8 T1
C11 T2
AAUT 2007-8 T2
C11 T3
AAUT 2007-8 T3
Fe accumulation in pigeon pea seeds in different varieties
T1: Control; T2: 20 kg/ha FeSO4 soil application; T3: Soil Fe application along with 0.5%
FeSO4 foliar application.
ACKNOWLEDGEMENTS
The author is grateful to Centre of Excellence (Ref No. 1205 / saater – 4 - 2013, dated 3.1.14
for the financial assistance.
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Corresponding author: Dr. Rajni Shukla, ICAR-NAIP, Department of Botany, Lucknow
University, Lucknow- 226 007, U.P., India
Email: rajni_shukla1@yahoo.co.in yogesh_s26@yahoo.com
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