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Saleem et al. BMC Plant Biology (2023) 23:397
https://doi.org/10.1186/s12870-023-04400-x BMC Plant Biology
*Correspondence:
Asma Zulqar
asma.botany@pu.edu.pk
Mehdi Rahimi
mehdi83ra@yahoo.com
Full list of author information is available at the end of the article
Abstract
Agricultural production is severely limited by an iron deciency. Alkaline soils increase iron deciency in rice
crops, consequently leading to nutrient deciencies in humans. Adding iron to rice enhances both its elemental
composition and the nutritional value it oers humans through the food chain. The purpose of the current
pot experiment was to investigate the impact of Fe treatment in alkaline (pH 7.5) and acidic (pH 5.5) soils to
introduce iron-rich rice. Iron was applied to the plants in the soil in the form of an aqueous solution of FeSO4
with ve dierent concentrations (100, 200, 300, 400, and 500 mM). The results obtained from the current study
demonstrated a signicant increase in Fe content in Oryza sativa with the application of iron in both alkaline and
acidic pH soils. Specically, Basmati-515, one of the rice cultivars tested, exhibited a notable 13% increase in iron
total accumulation per plant and an 11% increase in root-to-shoot ratio in acidic soil. In contrast to Basmati-198,
which demonstrated maximum response in alkaline soil, Basmati-515 exhibited notable increases in all parameters,
including a 31% increase in dry weight, 16% increase in total chlorophyll content, an 11% increase in CAT
(catalase) activity, 7% increase in APX (ascorbate peroxidase) activity, 26% increase in POD (peroxidase) activity,
and a remarkable 92% increase in SOD (superoxide dismutase) in acidic soil. In alkaline soil, Basmati-198 exhibited
respective decreases of 40% and 39% in MDA and H2O2 content, whereas Basmati-515 demonstrated a more
signicant decrease of 50% and 67% in MDA and H2O2 in acidic soil. These results emphasize the potential for
targeted soil management strategies to improve iron nutrition and address iron deciency in agricultural systems.
By considering soil conditions, it is possible to enhance iron content and promote its availability in alkaline and
acidic soils, ultimately contributing to improved crop nutrition and human health.
Keywords Iron biofortication, Iron fertlizer, Soil fertility, Soil pH, Fe accumulation
Alkaline and acidic soil constraints on iron
accumulation by Rice cultivars in relation
to several physio-biochemical parameters
AmmaraSaleem1, AsmaZulqar1*, Muhammad ZafarSaleem2, BaberAli3, Muhammad HamzahSaleem4,
ShafaqatAli5,6, Ebru DerelliTufekci7, Ali RızaTufekci8, MehdiRahimi9* and Reham M.Mostafa10
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Saleem et al. BMC Plant Biology (2023) 23:397
Introduction
Rice is the best species for genetic characterization of
Iron (Fe) homeostasis and genetic improvement since it
is the most adaptable model for cereals and a food crop
with economic importance [1]. Two thirds of people are
fed by rice, a basic staple diet. In developing nations, it is
essential for ensuring food security [2]. In order to reach
Zero Hunger, hidden hunger must be tackled as one of
the primary diculties, especially in African, Asian, and
Latin American nations where it aects more than two
billion people. In fact, almost three million people die
each year from nutritional deciencies, primarily due to a
deciency of vitamins and minerals [3].
Numerous vital activities, including as development,
cognition, the immune system, and maintaining antioxi-
dant activity, depend on micronutrients like Zn, Fe, and
Se [4]. Fe plays a critical role in the catalytic activities
of numerous enzymes, including those involved in the
transport of oxygen, the transfer of electrons, oxi-reduc-
tion events, collagen manufacture, and the metabolism of
vitamin D [5]. Anemia, which is dened as having inad-
equate red blood cells, accounts for a signicant num-
ber of cases [6]. Children and adults with chronic IDA
(Iron Deciency Anemia) experience substantial growth
and developmental problems, including delayed growth,
which results in weariness and lowers physical and men-
tal function [7].
e fourth most prevalent element in the earth’s crust
is iron (Fe), and it is crucial for both chemical and bio-
logical activities. Recent decades have seen an increase
in interest in environmental chemistry and material
sciences about the redox reactivity of diverse forms of
Fe [8]. Fe is an essential component of many enzymes
and proteins in vital processes that support growth and
metabolism in plants, and a lack of it is directly linked to
a decline in crop production and quality because it is cru-
cial for plant growth, productivity, and quality. Algae and
higher plants’ photosynthetic eciency is signicantly
inuenced by Fe homeostasis [9]. Fe has an impact on the
cycling of nitrogen (N) in soils in both oxic and anoxic
conditions. e pH of the soil controls how much Fe is
used in the conversion of N. Fe oxides frequently stimu-
late nitrication activity in soil with low pH, where their
impact on soil N transformation activities is dependent
on soil pH [10].
e importance of iron in rice accumulation is attrib-
uted to its role in the synthesis and functioning of pro-
teins and enzymes involved in the transport and storage
of nutrients. Iron is particularly important in the syn-
thesis of iron-containing proteins, such as ferredox-
ins, which are essential for electron transfer reactions
in photosynthesis and respiration. ese processes are
critical for energy production and metabolism within the
plant [11, 12]. Iron deciency in rice occurs due to poor
solubility in ooded soils., Alkaline or high pH, excessive
phosphorus, and other nutrients [13, 14].
One of the main environmental factors that hinders
plant development and yield production globally is alka-
line stress. Alkaline or high pH soils negatively aect
iron availability. In such conditions, iron tends to form
insoluble compounds, making it dicult for rice plants
to take up sucient iron Additionally, alkaline stress
caused oxidative damage in plants that was reected in
greater levels of superoxide radical (O2•), hydrogen per-
oxide (H2O2), methylglyoxal (MG), and malondialdehyde
(MDA) [15]. On alkaline soils, plants may exhibit iron
deciency chlorosis (IDC), which inhibits development
and yield [16]. Alkaline (high pH) soils may also inhibit
the absorption of other metal micronutrients such as Mn
and Zn [17]. e concentration of carbonate (CO32−) and
bicarbonate (HCO3) increases with a rise in the pH of the
growth medium, while the solubility of iron decreases as
a result of H+ being consumed by HCO3 [18]. Numerous
research oer specic solutions to address this issue and
improve the micronutrient content in crops [19, 20].
More than 50% of the world’s population is fed on rice
(O. sativa L.). One of the most essential staple crops is
rice, but polished rice has a low quantity of important
micronutrients, various strategies such as biofortica-
tion are useful to cope with this problem and increase
the concentration of micronutrients in crops [21, 22].
As a result, the major purpose of current research work
is to enhance the content of iron in four O. sativa “culti-
vars: Basmati-198, Basmati-515, PK-386, and KSK-133”. It
was also determined how dierent iron treatments alter
morphological, physiological, antioxidant defense sys-
tems, and iron absorption. For this purpose, a study was
directed to investigate the response of O. sativa to vari-
ous concentrations of FeSO4 at two pH levels (alkaline
and acidic), as well the suitable Fe concentration and pH
for normal rice growth and development in iron decient
soil.
Materials and methods
Experimental plan
Seeds of rice (Oryza sativa L.) were received from the
Rice Research Institute, KSK, and the rice varieties used
in the present research work were “Basmati-198, Bas-
mati-515, PK-386, and KSK-133”. Two Basmati varieties
were selected in present research work According to our
earlier research, the iron concentrations of Basmati-515
and Basmati-198, are 22.0 and 14.1 ppm, respectively,
whereas PK-386 has 19.0 ppm [23]. Seeds of four rice cul-
tivars surface sterilized by using 0.1% bleaching powder
(10 to 20min), then gently rinsed with deionized water
before being planted in plastic pots in a natural environ-
ment condition (day temperature: 36 oC and night tem-
perature: 27 oC).
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Saleem et al. BMC Plant Biology (2023) 23:397
Soil preparation
e garden soil (silt loam) from the eld of the Botanical
Garden, University of the Punjab, Lahore (31o29’57.78N
latitude and 74o17’58.60 E longitude) was collected. e
soil samples were air-dried, and their physico-chemical
properties are presented (Table1).
roughout the experiment, the pH of the soil for each
treatment used in this study was adjusted to 5.5 for acidic
soil and 7.5 for alkaline soil, respectively. In this experi-
ment, a randomized complete block design (RCBD) with
six replications was used, and iron sulfate (FeSO4) was
manually mixed to achieve the ve treatment concentra-
tions (100, 200, 300, 400, and 500 mM) along with the
control. e control plants were left untreated, without
the addition of any specic treatments.
Morphological parameters
e plants were harvested on July 16, 2022, after 25 days
of treatment, for the dierent morphological and bio-
chemical parameters. To eliminate dirt and waste, the
plants from each treatment were rst washed with tap
water and then with distilled water. After the plants were
harvested, morphological measurements including total
plant length, shoot fresh weight, root fresh weight, and
total dry weight per plant were recorded in alkaline as
well as acidic soil.
Iron accumulation in plant
Samples of Fe-added rice varieties were collected to
assess the Fe concentration in their tissues. Whole plants
washed with distilled water after harvesting to remove
soil, and either after all these plants kept in cold DCB
(dithionite-citrate-bicarbonate) solution for 3h for iron
content determination. Samples were heated to 500°C
for 3h and then dried at 60 °C. By using concentrated
HCl to decompose the ashes, iron was then measured
using atomic absorption spectrophotometry [24].
Estimation of antioxidant enzyme activities
A leaf of fresh plant material weighing 0.2g was taken
and homogenized in phosphate buer (5 mL) and liquid
nitrogen having a pH of neutral. At 4°C and 12,000rpm,
the homogenate mixture was centrifuged for 20min, and
then the extracts were stored at -20°C while the superna-
tants were discarded.
e antioxidant enzymes CAT [25], APX [26], POD
[27], SOD [28], GPX [29] and DPPH [30] were measured
at absorbance of 550, 290, 420, 405, 460, 460, and 517nm
respectively.
Estimation of oxidative stress markers
A mixture of 3 mL of sample extract and 1 mL of 0.1%
titanium sulphate in 20% (v/v) H2SO4 was centrifuged
at 6000g for 15min to determine the H2O2 content of
plant tissues. e yellow colour intensity was measured
at 410nm [31]. According to [32], the quantity of MDA
was determined.
Results
Eect of Fe on morphology of O. sativa cultivars in soil (pH
7.5 and pH 5.5)
After 25 days of treatment, plants of all O. sativa culti-
vars planted in high doses of Fe amended soils displayed
a better plant growth with higher height and weight.
Compared to control plants, O. sativa cultivars exhibited
dierent growth trends at varying FeSO4 concentrations.
In Fig. 1 (A, B), displayed the various morphological
characteristics of the O. sativa cultivars. Findings of the
study showed that plant height, root fresh weight, shoot
fresh weight, and total dry weight per plant increased by
increasing Fe concentrations (100, 200, 300, 400, and 500
mM) than those of control plants. Results from the cur-
rent study also indicated that at all Fe levels, acidic soil
(pH 5.5) produced more signicant eects from Fe treat-
ment than alkaline soil with a pH of 7.5. At 500 mM Fe
treatments, the height of Basmati-198 and Basmati-515
increased in comparison to control plants by 47% and
45%, respectively (Fig. 1A) in soil having pH alkaline
(pH 7.5). Exogenous Fe supply in acidic soil dramatically
increased plants height as compared to plants grown in
alkaline soil. Basmati-515 in acidic soil showed maximum
plant height results that were 63% higher than the con-
trol and Basmati-198 in alkaline soil (Fig.1B). In alkaline
soil, Basmati-198 exhibited a substantial increase in root,
shoot fresh weight, and dry weight, with respective incre-
ments of 94%, 71%, and 491% compared to untreated
plants. Additionally, when compared to Basmati-198,
Basmati-515 displayed a 285% and 6% increase in root
and shoot fresh weight, respectively, and a remarkable
31% increase in total dry weight in acidic soil. ese
ndings underscore the contrasting growth responses
between the two cultivars, indicating the superior perfor-
mance of Basmati-515 in terms of root and shoot fresh
weight and total dry weight. In control plants, total dry
Table 1 The physico-chemical properties of soil samples
Properties Acidic soil Alkaline soil
Depth (cm) 0–12 0–12
pH 5.5 7.5
Electrical Conductivity (dS/cm) 0.7 0.7
Organic matter (%) 0.19 0.19
Available phosphorus (mg/kg) 2.98 2.43
Available potassium (mg/kg) 66.4 52.4
Available nitrogen (mg/kg) 94 32
Iron (mg/kg) 67 51
Saturation (%) 25% 25%
Texture Silty loam Silty loam
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Saleem et al. BMC Plant Biology (2023) 23:397
weight was signicantly lower as compared to Fe-treated
plants (Fig.1C- H).
Eect of Fe on photosynthetic parameters of O. sativa
cultivars in soil (pH 7.5 and pH 5.5)
e chlorophyll and carotenoids analysis revealed that
application of FeSO4 enhanced chlorophyll and carot-
enoids content in O. sativa. e results regarding chloro-
phyll and carotenoids (chl-a, chl-b, and total chlorophyll
content) in alkaline and acidic soil under FeSO4 treat-
ment are presented in Fig. 2. Chlorophyll-a increased
by 85 and 65% in alkaline and acidic soils, respectively,
in O. sativa cultivar (Basmati-198) in contrast to control.
While the highest chl-a was observed in acidic soil, it was
observed in Basmati-515 with an average value of 2.8mg
kg− 1 FW at the highest concentration 500 mM in com-
parison to the control with a value of 1.8mg kg− 1 FW
(Fig.2A, B). e chl-b content was signicantly increased
with the same pattern as in chl-a, but in comparison,
the chl-a content was higher at each concentration in
all cultivars (Figure C, D). Total chlorophyll content was
strongly correlated with chl-a and chl-b content. e
results regarding this showed that total chlorophyll con-
tent was signicantly high in Basmati-515, with values
of 3.28 and 4.2mg kg− 1 FW in alkaline and acidic soils,
respectively. In contrast to Basmati-198, which exhib-
ited maximum results in alkaline soil, Basmati-515 dem-
onstrated a signicant 16% increase in total chlorophyll
content specically in acidic soil (Figure E, F). We also
discovered that raising the concentrations of FeSO4 in the
soil caused a substantial (p < 0.05) rise in the carotenoids
content in alkaline as well as acidic soil (Figure G, H).
Eect of Fe on Fe accumulation in shoot, root, total
accumulation in plants and root-to-shoot Fe ratio of O.
sativa cultivars in soil (pH 7.5 and pH 5.5)
We assessed another important eect of application of
FeSO4 on iron accumulation in shoots, roots, and total
Fe accumulation in plants, as well as root-to-shoot Fe
ratio (Fig.3). is showed that by increasing the levels of
FeSO4 contents, iron accumulation was also increased.
As compared to the control plants, iron accumula-
tion increased in all cultivars of O. sativa under the dif-
ferent treatments of Fe. Basmati-198 accumulated the
Fig. 1 Eect of Fe (0, 100, 200, 300, 400, and 500 mM) on selected O. sativa cultivars under alkaline and acidic pH. Plant height (A, B), root fresh weight
(C, D), shoot fresh weight (E, F), and total dry weight per plant (G, H). According to DMRT, bars with distinct alphabet letters are substantially (p < 0.05)
dierent from one another. All data in the graph are the averages of six replicates (n = 6)
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Saleem et al. BMC Plant Biology (2023) 23:397
most Fe in alkaline soil, while Basmati-515 accumulated
the higher Fe in acidic soil (Fig.3). e root-to-shoot Fe
ratio was also elucidated, with data demonstrating that
increasing FeSO4 concentrations resulted in a substantial
(p < 0.05) rise in Fe contents of O. sativa cultivars when
compared to control (Fig.3). Iron accumulation in the
shoot of the O. sativa cultivar Basmati-198 exhibited
a signicant increase of 175% in alkaline soil and 117%
in acidic soil compared to the respective control condi-
tions (Fig.3A, B). However, all other parameters, includ-
ing iron accumulation in the root, total accumulation,
and root to shoot ratio, showed a signicant positive
response towards the highest concentrations of FeSO4 in
alkaline and acidic soil (Fig.3C–H). Upon further analy-
sis, it was found that Basmati-515 exhibited the high-
est iron accumulation in roots when treated with a high
concentration of FeSO4 (500 mM), with an average value
of 350 root accumulation P− 1. Notably, Basmati-515 dis-
played an 18% increase in iron accumulation in acidic soil
compared to Basmati-198, which exhibited its highest
response in alkaline soil with a value of 287 root accumu-
lation P− 1 (Fig.3C, D). e O. sativa cultivar Basmati-198
demonstrated the highest total iron accumulation in alka-
line soil, while Basmati-515 displayed a more favourable
response in acidic soil, reaching a maximum total accu-
mulation of 630 and 555 total accumulation P− 1, respec-
tively at 500 mM. Furthermore, when comparing the two
Basmati cultivars, Basmati-515 exhibited a notable 13%
increase in total accumulation in acidic soil (Fig.3E, F).
Root to shoot iron accumulation ratio was increased in
the same pattern as iron accumulation in leaves, roots,
and total accumulation in plants. In terms of root-to-
shoot iron accumulation, Basmati-198 exhibited an aver-
age value of 2.6 in treated plants in alkaline soil. On the
other hand, Basmati-515 displayed a higher average value
of 2.9 in acidic soil compared to untreated plants. When
comparing the two cultivars, Basmati-515 demonstrated
signicantly higher results, with an 11% increase in iron
accumulation, specically in acidic soil. is indicates the
stronger responsiveness of Basmati-515 to iron treatment
in an acidic soil environment. (Fig.3G, H).
Fig. 2 Eect of Fe (0, 100, 200, 300, 400, and 500 mM) on selected O. sativa cultivars under alkaline and acidic pH. Chlorophyll a (A, B), chlorophyll b (C,
D), total chlorophyll content (E, F), and carotenoids (G, H). According to DMRT, bars with distinct alphabet letters are substantially (p < 0.05) dierent from
one another. All data in the graph are the averages of six replicates (n = 6)
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Saleem et al. BMC Plant Biology (2023) 23:397
Eect of Fe on antioxidant enzymatic activity (CAT, APX,
POD and SOD) of O. sativa cultivars in soil (pH 7.5 and pH
5.5)
e data related to the antioxidant enzymatic activ-
ity (CAT, APX, POD and SOD) of O. sativa cultivars in
alkaline (pH 7.5) and acidic soil (pH 5.5) as depicted in
Fig.4. e treatment of dierent doses of iron has a posi-
tive eect on all antioxidant enzymes and by increasing
the concentration of FeSO4 signicantly high results were
obtained in Basmati-515. Maximum antioxidant enzy-
matic activity was observed in Basmati-198 in alkaline
soil, while Basmati-515 showed the maximum antioxi-
dants in acidic soil (Fig.4A-H). A dose of 500 mM FeSO4
induced a signicant increase in CAT activity in alkaline
and acidic soils, with values of 0.43 and 0.48 UgP− 1 in
Basmati-198 and Basmati-515, respectively. Basmati-198
displayed a notable 69% increase in APX (ascorbate per-
oxidase) activity in alkaline soil, whereas Basmati-515
exhibited maximum results in acidic soil with a 62%
increase in APX activity Fig.4 (C, D). Basmati-198 dem-
onstrated a signicant 55% increase in POD activity in
alkaline soil, while Basmati-515 displayed the highest
results with a remarkable 65% increase in POD activity
specically in acidic soil (Fig.4E, F). Basmati-515 showed
a maximum SOD value at a level of 400 and 500 mM
of FeSO4, with an average value of 0.41 and 0.52 UgP− 1
higher than the control, which has a value of 0.31 UgP− 1.
Basmati-515 exhibited a substantial 95% higher increase
in acidic soil compared to Basmati-198 (Fig.4G, H). is
highlights the superior responsiveness of Basmati-515 in
terms of the observed increase in all antioxidants.
Eect of Fe on antioxidant enzymatic activity (GPX and
DPPH) and oxidative markers (MDA and H2O2) of O. sativa
cultivars in soil (pH 7.5 and pH 5.5)
Figure5 depicts another important eect of the applica-
tion of Fe on the antioxidant enzymes and total antioxi-
dant activity (GPX and DPPH), as well as the reduction
in oxidative stress markers (MDA and H2O2) in O. sativa.
e results showed that, by increasing the application
treatment of FeSO4, GPX and DPPH increased, as in the
case of the other antioxidant enzymes in Fig.4. As com-
pared to the control plants, GPX and DPPH increased in
all cultivars of O. sativa under the dierent treatments of
Fig. 3 Eect of Fe (0, 100, 200, 300, 400, and 500 mM) on selected O. sativa cultivars under alkaline and acidic pH. Fe accumulation in shoot (A, B), Fe
accumulation in roots (C, D), Fe total accumulation in plant (E, F), and root to shoot Fe ratio (G, H). According to DMRT, bars with distinct alphabet letters
are substantially (p < 0.05) dierent from one another. All data in the graph are the averages of six replicates (n = 6)
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Saleem et al. BMC Plant Biology (2023) 23:397
Fe. GPX activity was high in Basmati-198 in alkaline soil,
while in acidic soil, Basmati-515 showed a signicant high
result with a value of 0.07 and 0.085 UgP− 1, respectively
(Fig.5A, B). When compared to the control in acidic soil
at 80%, increasing the treatment level of FeSO4 in the soil
resulted in a substantial (p < 0.05) rise in the DPPH of
O. sativa cultivars (Fig.5D). In contrast to antioxidants,
oxidative stress markers (MDA and H2O2) decreased
in all O. sativa cultivars. Basmati-198 exhibited a 50%
decrease in MDA (malondialdehyde) content in alkaline
soil, while Basmati-515 showed a 40% decrease in MDA
in acidic soil. Comparatively, Basmati-515 displayed a 9%
greater decrease in MDA in acidic soil compared to Bas-
mati-198. (Fig.5E, F). In terms of H2O2 (hydrogen perox-
ide) content, Basmati-198 demonstrated a 39% decrease
in alkaline soil, whereas Basmati-515 exhibited a more
substantial 67% decrease in acidic soil when compared
to untreated plants. Furthermore, in alkaline soil, Bas-
mati-515 displayed a 54% greater decrease in H2O2 con-
tent compared to Basmati-198 (Fig.5G, H). e oxidative
stress levels were signicantly lower in acidic soil com-
pared to all treated and untreated plants in alkaline soil.
Eect of Fe on soluble sugar, avonoids, free amino acids,
and carbohydrates of O. sativa cultivars in soil (pH 7.5 and
pH 5.5)
e eect of application of FeSO4 on antioxidants non-
enzymatic activities (soluble sugar, avonoids, total free
amino acids, and carbohydrates) in O. sativa cultivars is
presented in Fig. 6. By increasing the concentration of
FeSO4, all non-enzymatic activities were increased, in
the case of the antioxidant enzymes as compared to the
control (Fig. 4). Soluble sugar was signicantly higher
in Basmati-198 in alkaline soil, while in acidic soil, Bas-
mati-515 showed a signicant high result with a value
of 48.8 and 61mg g− 1 FW, respectively (Fig.6A, B). e
application of increased FeSO4 concentrations in alka-
line soil resulted in a statistically signicant improve-
ment in avonoid content in Basmati-198, compared
to the control (0.31 µmol g-1 FW). Notably, the highest
avonoid content was observed in Basmati-515, with an
average value of 0.5 µmol g-1 FW. ese ndings, dem-
onstrate the potential of FeSO4 treatment in promoting
avonoid accumulation, with Basmati-515 displaying a
better response in terms of avonoid content (Fig.6 C,
Fig. 4 Eect of Fe (0, 100, 200, 300, 400, and 500 mM) on selected O. sativa cultivars under alkaline and acidic pH. CAT activity (A, B), APX activity (C, D),
POD activity (E, F), and SOD activity (G, H). According to DMRT, bars with distinct alphabet letters are substantially (p < 0.05) dierent from one another.
All data in the graph are the averages of six replicates (n = 6)
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Saleem et al. BMC Plant Biology (2023) 23:397
D). Basmati-198 exhibited a substantial 76% increase in
free amino acid content, while Basmati-515 displayed
an even greater 80% increase. ese results indicate the
potential of both Basmati-198 and Basmati-515 varieties
to enhance the accumulation of free amino acids when
treated with FeSO4 (Fig.6E, F). In a comparison between
Basmati-515 and Basmati-198, Basmati-515 exhibited a
7% higher increase in carbohydrate content in acidic soil
compared to Basmati-198 in alkaline soil. is suggests
that Basmati-515 is more responsive to acidic soil condi-
tions in terms of carbohydrate accumulation (Fig.6G, H).
Discussion
Iron is required for a variety of metabolic activities in
plants. However, its low availability in high pH soils and
roots’ reduced ability to acquire it due to iron are two of
the most important problems restricting plant develop-
ment [33]. Micronutrient deciencies, encompassing
calcium, selenium, zinc, and iron, are prevalent among a
signicant portion of the global population. ese de-
ciencies contribute to malnutrition and pose a substantial
burden on public health worldwide [34]. Zinc (Zn) and
Iron (Fe) are the two most commonly decient micro-
nutrients, and they both play vital roles in supporting
overall health and well-being [35]. Alkaline soils dem-
onstrate a lack of micronutrients on a global scale [36].
e required levels of micronutrients vary depending
on the soil type and pH, necessitating dierent nutrient
considerations for optimal plant growth, and develop-
ment [37]. pH of the soil is a key component that inu-
ences nutrient availability for plants, and soil in arid areas
is generally alkaline with a high pH [38]. Despite the
widespread recognition of chlorosis occurring in alkaline
soils with limited iron (Fe) availability, the antioxidant
and physiological responses to iron deciency remain
poorly characterized. ere is a lack of comprehensive
understanding regarding the specic mechanisms and
responses associated with iron deciency, highlighting
the need for further research in this area. Exploring the
antioxidant and physiological dynamics under iron-de-
cient conditions can contribute to a more comprehensive
understanding of the impact of iron deciency on plant
health and provide insights into potential strategies for
mitigating the detrimental eects of iron deciency in
Fig. 5 Eect of Fe (0, 100, 200, 300, 400, and 500 mM) on increasing the level of antioxidants (GPX (A, B); DPPH (C, D)) and reducing oxidative stress
levels (MDA (E, F); H2O2 (G, H)) in selected Oryza sativa cultivars under alkaline and acidic pH. According to DMRT, bars with distinct alphabet letters are
substantially (p < 0.05) dierent from one another. All data in the graph are the averages of six replicates (n = 6)
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Saleem et al. BMC Plant Biology (2023) 23:397
alkaline soil environments. In this study, we examined the
eects of pH and iron (Fe) supply as two factors on plant
responses. Our ndings revealed that plant responses
to Fe supply were signicant in acidic soil pH but not in
alkaline pH. e experiment involved ve Fe treatments
at pH levels of 5.5 and 7.5. O. sativa cultivars grown in
soil treated with FeSO4 exhibited enhanced plant growth,
improved antioxidant activity, and increased iron accu-
mulation. ese results emphasize the importance of
considering both soil pH and iron supplementation in
optimizing plant performance and nutrient uptake in rice
cultivation.
Fe is a crucial micronutrient that plays a signicant role
in numerous biological processes and is essential for both
human health and plant vitality [39]. In alkaline soils with
a pH ranging from 7.4 to 8.5, iron minerals tend to have
low solubility and slow dissolution kinetics. e uptake of
iron by plants growing in alkaline soils is further hindered
by elevated bicarbonate levels, a characteristic feature of
calcareous soils. As a result, the concentration of available
iron becomes inadequate for optimal plant growth, lead-
ing to common occurrences of iron deciency in plants
[40]. Fe is necessary for the plants growth, and devel-
opment. While iron is present in abundance in soil, the
fraction of iron that is readily available for plant uptake
is often limited. is limited availability of accessible iron
in the soil can pose a challenge for plants to meet their
iron requirements, potentially leading to iron deciency
[41]. O. sativa cultivars grown in soil treated with FeSO4
(100, 200, 300, 400 and 500 mM), both in acidic and alka-
line soil conditions, exhibited notable improvements in
various morphological characteristics, including growth,
plant height, and fresh and dry mass. Iron can contrib-
ute to increased plant height. It is a crucial component of
various enzymes involved in protein synthesis. Proteins
are essential for cell division and elongation, which con-
tribute to overall plant growth and height. Adequate iron
levels ensure the proper functioning of these enzymes,
facilitating optimal protein synthesis and promoting
plant height development [42]. Our study, which focused
on the impact of FeSO4 on plant growth and morphol-
ogy, is highly relevant to the ndings from the previous
study regarding the role of iron in plant hormone regula-
tion and cell elongation. e previous study highlighted
Fig. 6 Eect of Fe (0, 100, 200, 300, 400, and 500 mM) on selected O. sativa cultivars under alkaline and acidic pH. Soluble sugar (A, B), avonoids (C, D),
free amino acids (E, F), and total carbohydrates (G, H). According to DMRT, bars with distinct alphabet letters are substantially (p < 0.05) dierent from one
another. All data in the graph are the averages of six replicates (n = 6)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 13
Saleem et al. BMC Plant Biology (2023) 23:397
that iron is involved in the biosynthesis and regulation
of plant hormones, specically auxins. Iron inuences
the synthesis and distribution of auxins, which, in turn,
impacts cell elongation and ultimately contributes to
increased plant height [43]. By ensuring sucient iron
availability through FeSO4 treatment, our study dem-
onstrated the positive impact of iron on morphological
characteristics in O. sativa cultivars. e increased iron
availability stimulated chlorophyll synthesis, enzymatic
activity, and hormone regulation, all of which collectively
contributed to improved growth, plant height, and bio-
mass accumulation. e lack of comprehensive studies
and understanding regarding the uptake and transloca-
tion of iron from the soil has hindered the development
of rice cultivars. However, previous research has shown
that for cultivars such as BARI-2000 and BARD-699,
foliar applications of iron treatments led to growth and
photosynthetic rate increases of up to 58% and 70%,
respectively. Furthermore, Various morpho-physiological
parameters, including shoot length, root length, shoot
fresh and dry weights, root fresh and dry weights, photo-
synthetic and transpiration rates, as well as SPAD values,
were also observed to increase [44].
Signicant decreases in plant height, shoot fresh
weight, root dry weight, germination percentages, and
photosynthesis (chl a, chl b, total chlorophyll, and carot-
enoids content) were observed in iron-decient rice
cultivars in alkaline as well as acidic soil (Figs.1 and 2).
Previous studies that examined morphological traits
under dierent iron levels and chlorophyll contents have
shown that iron deciency can lead to chloroplast degen-
eration in plant leaves [45]. Salinity refers to high levels
of salt in the soil, which can lead to osmotic stress as the
high salt levels disrupt water uptake and inhibit nutri-
ent absorption such as iron. Without sucient iron, the
photosynthetic rate of plants can be reduced, leading
to decreased shoot and root growth. Additionally, iron
deciency can aect the functioning of stomata, which
regulate gas exchange and transpiration rates. Reduced
stomatal conductance can further impact photosynthe-
sis and transpiration, limiting plant growth [46, 47]. Iron
deciency can disrupt electron transport and ATP syn-
thesis, leading to reduced energy availability for plant
growth and photosynthetic processes [48]. By increas-
ing the levels of FeSO4 contents, iron accumulation was
also increased. As compared to the control plants, iron
accumulation increased in all cultivars of O. sativa under
the dierent treatments of Fe (Fig.3). FeSO4 serves as a
source of soluble iron ions (Fe2+) that are readily available
for uptake by plant roots. As the concentration of FeSO4
increases, there is a higher concentration gradient of Fe2+
in the soil, which enhances the potential for iron uptake
by plant roots, leading to a higher accumulation of iron in
the plant tissues [12, 49].
In our current research, we observed signicant
enhancements in various antioxidants with increased
Fe treatment in both alkaline and acidic soil conditions
(Fig.4). ese ndings highlight the positive impact of
FeSO4 on multiple biochemical parameters associated
with plant health and stress response Specically, anti-
oxidants such as catalase (CAT), ascorbate peroxidase
(APX), peroxidase (POD), superoxide dismutase (SOD),
glutathione peroxidase (GPX), and the free radical scav-
enging capacity measured by the DPPH (2,2-diphenyl-
1-picrylhydrazyl) assay exhibited increased activity in
response to Fe treatment (Figs. 3 and 4). e positive
inuence of Fe supplementation on the plant’s antioxi-
dant capacity observed in our research can be related to
previous studies that showed the enhancement of anti-
oxidants due to the use of iron oxide nanoparticles [50].
Furthermore, Fe plays a role in regulating the expression
of genes involved in antioxidant defense. Fe availability
aects the transcription and translation of genes encod-
ing antioxidant enzymes, thereby increasing their pro-
duction and activity. is ultimately leads to an enhanced
antioxidant capacity in Fe-supplemented plants [51]. e
reduction in SOD, POD, and CAT activities in O. sativa
cultivars under signicant Fe deciency. Additionally, our
study claried that, under conditions of signicant iron
(Fe) deciency, the activities of key antioxidant enzymes,
reduced O. sativa cultivars reects the impact of Fe de-
ciency on the antioxidant defense system and increase in
oxidative stress (Fig.5). Our study is in line with the nd-
ings of previous research that investigated the exogenous
application of FeSO4 on O. sativa plants and its impact
on Cd toxicity, antioxidant potential, plant growth, and
photosynthesis. e previous study reported that the
application of FeSO4 resulted in decreased Cd toxicity by
enhancing the antioxidant potential and decreasing the
MDA and H2O2 level [52–54].
Furthermore, Fe treatment led to elevated levels of pro-
line, free amino acids, soluble sugars (including reduc-
ing and non-reducing sugars), avonoids, total soluble
protein, phenolic compounds, and total carbohydrates
(Fig.6). ese biochemical components are involved in
various physiological processes, such as osmotic adjust-
ment, stress tolerance, defense mechanisms, and energy
metabolism. By establishing a connection between our
research and previous studies, it is reported that Fe is
known to interact with other components of the anti-
oxidant system, such as non-enzymatic antioxidants like
phenolic compounds and avonoids, further enhancing
the antioxidant capacity of the plant [55]. According to
our study, increasing the iron concentration level in the
soil can lead to an increase in free amino acids and car-
bohydrates and enhance the uptake of iron by plants.
ese ndings are in agreement with previous studies.
Iron levels lead to increased carbohydrate production in
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 11 of 13
Saleem et al. BMC Plant Biology (2023) 23:397
plants, strengthen cell walls, and provide building blocks
for the synthesis of defense-related compounds [56].
Another study showed that iron is essential for maintain-
ing optimal amino acid metabolism in plants [57, 58]. A
signicant increase in avonoid content in O. sativa cul-
tivars was caused by increased FeSO4 concentrations in
comparison to the control (0.27 µmol g− 1 FW) in acidic
soil at 500 mM (Fig.6D). In addition to acting as antioxi-
dants, avonoids also contribute to plant metal tolerance
by donating hydrogen atoms. It has been demonstrated
that plants are more tolerant of stress when their avo-
noids are higher [59].
It was observed in the present study that iron availabil-
ity in the soil is highly inuenced by soil pH. In alkaline
soils, iron tends to form insoluble compounds, making
it less available for plant uptake. However, Fe treatment
enables them to better access and acquire iron in alkaline
soil. On the other hand, in acidic soils, iron is generally
more soluble and available for plant uptake; therefore, O.
sativa cultivars adapted to acidic soil conditions under Fe
treatment eciently take up and accumulate iron. Fur-
thermore, the eciency of iron uptake and transport is
closely related to the characteristics of the plant’s root
system, morphology, and antioxidants (Fig.7). Dierent
cultivars, such as Basmati-515 and Basmati-198, exhib-
ited variations in morphology and antioxidants that
inuenced the cultivar’s ability to acquire iron from the
soil, allowing them to accumulate higher iron levels in
either alkaline or acidic soil conditions. Further research
is needed to explore the genetic and physiological basis
for the dierential iron accumulation observed in various
cultivars and soil pH conditions.
Conclusion
On the basis of current ndings, it can be concluded that
Fe deciency caused a signicant decrease in all mor-
phological characteristics such as plant height, root and
shoot fresh and dry biomass, total plant biomass pro-
duction, photosynthetic activity, and gas parameters due
to the lower availability of Fe in alkaline soil. While sig-
nicant increases in oxidative stress markers (MDA and
H2O2) increased in Fe decient conditions, which were
reduced by treatment with FeSO4 by the production of
CAT, APX, POD, SOD, GPX and DPPH. us, the appli-
cation of Fe is a safer and better way to increase iron
content in O. sativa as well as biomass production. Bas-
mati-515 and Basmati-198 performed better in alkaline
and acidic soils, respectively, than the control and other
cultivars of O. sativa. However, the acidic and alkaline
restrictions on iron availability in soil, as well as the inu-
ence of pH on iron transporter genes, must be investi-
gated further at the molecular level.
Acknowledgements
Not applicable.
Authors’ contributions
Conceptualization, Ammara Saleem, Asma Zulqar; Muhammad Zafar
Saleem; Data curation, Baber Ali, Muhammad Hamzah Saleem, Reham M.
Mostafa; Formal analysis, Shafaqat Ali, Ebru Derelli Tufekci, Ali Rıza Tufekci,
Reham M. Mostafa, Baber Ali, Muhammad Hamzah Saleem; Investigation,
Ammara Saleem, Asma Zulqar; Methodology; Muhammad Zafar Saleem;
Project administration, Asma Zulqar; Resources, Asma Zulqar; Software,
Muhammad Hamzah Saleem and Baber Ali; Validation, Shafaqat Ali;
Visualization, Mehdi Rahimi., Shafaqat Ali; Writing – original draft Ammara
Saleem, Asma Zulqar; Muhammad Zafar Saleem; Writing – review & editing,
Ammara Saleem, Asma Zulqar; Muhammad Zafar Saleem, Baber Ali,
Muhammad Hamzah Saleem, Shafaqat Ali, Ebru Derelli Tufekci, Ali Rıza Tufekci,
Reham M. Mostafa, Mehdi Rahimi.
Fig. 7 Impact of iron fertilizer treatment on iron accumulation in rice plants and pH-dependent solubility
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 12 of 13
Saleem et al. BMC Plant Biology (2023) 23:397
Funding
Not applicable.
Data Availability
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
In this study, experimental research, and eld studies on plants (either
cultivated or wild), including the collection of plant material involved from
University of Agriculture, Multan, Pakistan. All the protocols and experiment
were conducted according to national, and international guidelines and
legislation.
Consent for publication
Not applicable.
Conict of Interest
The authors declare that the research was conducted in the absence of any
commercial or nancial relationships that could be construed as a potential
conict of interest.
Competing interests
The authors declare no competing interests.
Author details
1Institute of Botany, University of the Punjab Lahore, Lahore
54590, Pakistan
2Centre for Applied Molecular Biology, University of the Punjab Lahore,
Lahore 54590, Pakistan
3Department of Plant Sciences, Quaid-i-Azam University,
Islamabad 45320, Pakistan
4College of Plant Science and Technology, Huazhong Agricultural
University, Wuhan 430070, China
5Department of Environmental Sciences and Engineering, Government
College University, Faisalabad 38040, Pakistan
6Department of Biological Sciences and Technology, China Medical
University (CMU), Taichung City 40402, Taiwan
7Food and Agriculture Vocational School, Department of Field Crops,
Cankiri Karatekin Universitesi, 18100 Cankiri, Turkey
8Faculty of Science, Department of Chemistry, Cankiri Karatekin
Universitesi, Cankiri18100, Turkey
9Department of Biotechnology, Institute of Science and High Technology
and Environmental Sciences, Graduate University of Advanced
Technology, Kerman, Iran
10Department of Botany and Microbiology, Faculty of Science, Benha
University, Benha 13518, Egypt
Received: 9 June 2023 / Accepted: 3 August 2023
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