INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 856 www.rsisinternational.org

Physiological and Biochemical Attributes of Chromium

Detoxification are Regulated by Root synthesized Organic Acids

in Rice Varieties

Md. Habibur Rahman,

Md. Imran Hossain,

Md. Shadiqul Islam,

A.K.M. Nazmul Huda*

Dept. of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh

Dept. of Applied Chemistry and Chemical Engineering, Islamic University, Kushtia-7003,

Bangladesh

*Corresponding Author

DOI: https://dx.doi.org/10.51244/IJRSI.2025.1210000076

Received: 02 October 2025; Accepted: 08 October 2025; Published: 04 November 2025

ABSTRACT

To know effective physiological response against chromium toxicity, 25 rice varieties were cultivated

on the hydroponic solution treated with 100µM chromium and physiological as well as biochemical

features were evaluated compared with non-treated control plants. In this investigation, it was found that

the concentration of citric acid synthesized and secreted by roots influences the mitigation of chromium

toxicity. In varieties BR-58, BR-63 and BR-68 chromium uptake were significantly higher than the

control plant but their translocation to shoot was restricted indicating elevated Cr retention in roots. This

retention was facilitated by root secreted citric acid which was assured by significant rhizospheric pH

reduction (15%, 18.5% and 20.9% respectively) under chromium stress. Furthermore, BR-73 showed

an efficient exclusion mechanism keeping down metal uptake by citric acid ensured by 15% rhizosphere

pH reduction. In contrast, varieties of rhizospheres with a pH reduction of less than 10% were unable to

withstand chromium toxicity. The findings indicate that a reduction of 15% or more in rhizospheric pH

serves as the benchmark for the necessary level of organic acid secretion required for chromium

tolerance. Moreover, the strategies employed for tolerance differ based on genotypes rather than species.

Furthermore, it offers an efficient screening technique for metal tolerant rice plants.

Key word:

rhizospheric pH, chromium tolerant, organic acids, adsorption, Oryza sativa

INTRODUCTION

Chromium (Cr), a hazardous heavy component found in the outer layers of the Earth, has adverse effects

on the environment. It is thoroughly used in leather tanning, electroplating process, steel production,

metal finishing, catalyst uses and pigmentation. The relevant sources of Chromium (Cr) exposure in the

environment are industrial discharges and domestic sewage (Nath et al. 2008;). In plants, accumulated

Cr inhibits growth by limiting the absorption of nutrients (Ullah et al., 2023; Shanker et al., 2005). It

also causes chlorosis in young plants, lowers pigment content, alters enzymatic functions, damages root

cells, and induce ultrastructural changes to the cell membrane and chloroplast (Panda and Choudhury

2008). Cr also significantly impairs the development of stems and leaves during the early growth stage

of the plant, as well as the formation of dry matter in seedlings (Dey et al., 2023; Nematshahi et al.,

2012). The toxicity of Cr is dependent on the metal species that determine Cr's absorption, transport,

and accumulation. Numerous investigations on the chemistry of chromium in soil and its uptake by

plants have indicated that Cr is harmful for plant growth (Arun et al. 2005).

In order to withstand the toxicity of heavy metals, plants have an intricate and interrelated system of

defense mechanisms. Plant’s physical barriers, which include cell walls, physiologically active tissues

like trichomes, and morphological features like thick cuticle, are their first line of protection against

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 857 www.rsisinternational.org

metals (Al-Khayri et.al., 2023, Wong et.al., 2004, Harada et.al., 2010). To counteract and lessen the

negative effects of HMs, plants activate several cellular defense mechanisms when the metal ions

penetrate biophysical barriers and enter tissues and cells. Tolerating or neutralizing metal toxicity mostly

involves the biogenesis of several cellular macromolecules, including asnicotianamine, putrescine,

spermine, mugineic acids, organic acids, phytochelatins, metallothioneins, cellular exudates, heat shock

proteins, certain amino acids, and hormones (Viehweger 2014, Dalvi and Bhalerao 2013, Sharma and

Dietz 2006). Ineffectiveness of the aforementioned approaches in plants increases the production of

ROS (Mourato et.al. 2012). In order to eliminate the free radicals, plants then increase their enzymatic

antioxidants, which includes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX),

guaiacol peroxidase (GPX), and glutathione reductase (GR), as well as their nonenzymatic antioxidants,

such as ascorbate (AsA), glutathione (GSH), carotenoids, alkaloids, tocopherols, proline, and phenolic

compounds (Sharma et.al., 2012, Rastgoo et.al., 2011). However, genetic potential of plant species as

well as the metal type are the key determinant of metal tolerance level (Solanki and Dhankhar 2011).

Root excreted low molecular weight organic acid (LMWOA) also known to regulate different stress

especially oxidative stress (Airaki et al., 2012). Synthesized LMWOA released to the rhizosphere

through increased efflux (De La Fuente et al., 1997; López-Bucio et al., 2000a) but not authenticated

for every plant (RRRRR). Root excretion organic acid and its impact on physiology was studied enough

in previous. Most of this OA detoxify heavy metal in two ways. First one in the internal chelating of

heavy metals both in photosynthetic and non-photosynthetic tissues (Fernando et al., 2010). Second one

is the insoluble complex formation of organic acid with heavy metal and release phosphorus ion (Pi)

from the bound complex. Here positively charged cation of heavy metal react with negatively charged

carboxylic group of organic acids (OAs) to form insoluble complexes that not absorbed by plant (Zhang

et al., 2018).

However, these LMWOAs are synthesized in mitochondria and enzymes participate in glycolysis, TCA

and glyoxylate cycles are also responsible for the synthesis (Igamberdiev and Eprintsev, 2016). Stress

condition enhanced LMWOA synthesis by hampering the standard pathway of these cycles.

Furthermore, anaplerotic reactions (chemical reactions that form inter- mediates of the TCA cycle) rat

enhance OAs synthesis to cope with stress (Dong et al., 2004). Thus, higher level of transcription of

genes encoding TCA cycle enzymes as well as their enhanced enzymatic activities upregulate the

LMWOA synthesis and help plants to cope with metal toxicity (Zhou et al., 2018, Uhde-Stone et al.,

2003a).

In japonica rice, OsFRDL4 and OsFRDL2 are upregulated under Aluminum stress to secrete citric acid

from roots (Yokosho et al., 2011). But types and amount of root secreted organic acids are the lack of

clear evidence though it is assumed that surrounding environment may responsible for this specificity

(RRRRRR).

Consumption of carbon sources for production and efflux of OAs under stress utilize significant

proportion of carbon imposing an energy cost to plants which is economically important for plants

especially fast-growing annual crops (Koyama et al., 2000; Herz et al., 2018). Plants optimize its carbon

loss through tissue-specific or location specific exudation of OAs from the roots firstly. Secondly, it

limits the amount of OAs release by negative regulators of OA exudation like GABA (Ramesh et al.,

2015). But belowground rhizosphere deposition was not even considered in any models for carbon

allocation (Reyes et al., 2020). So, it demands future studies on this significant issue. Moreover, the

regulatory mechanisms regarding types and amount of root secreted organic acids remain largely

elusive.

However, effective physiological responses against toxic chromium in rice plants remain poorly

understood. These types of responses against metal toxicity are classified as either internal or external

(Kochian et al., 2004). Local rice variety, Pokkali develop tolerant against chromium by limiting its Fe

reductase and Fe transporter activities (Kabir, 2016). Furthermore, Zeng Fanrong et.al., (2008) reported

that rice plants released oxalic, malic, and citric acid at the rhizosphere and enhanced Cr accumulation.

But there are no reports on how organic acids work or if they have any effective role against chromium

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 858 www.rsisinternational.org

toxicity in rice. Moreover, external mechanisms, such as root exudation-mediated heavy metal

avoidance or tolerance, remain ill-defined. Therefore, different high-yielding rice varieties were exposed

to Cr stress and evaluated the physiological and biochemical features to gain new insights into metal

tolerance in rice.

MATERIALS AND METHODS

Plant cultivation

In this study, seeds from 25 authentic rice varieties (Bangladesh Rice Research Institute variety BRRI

50 to 73 and 22) were initially collected at the germplasm center of the Bangladesh Rice Research

Institute. After being sterilized with 95% (v/v) ethanol, water-washed seeds were allowed to germinate

in wet filter paper Petri dishes for two to three days at room temperature in the dark. The plants that

germinated uniformly were transferred to the Hoagland and Arnon (1950) hydroponic solution (100 ml).

For rice seedlings under chromium stress, a hydroponic solution containing 100 µM K2Cr2O7 (a source

of the heavy metal chromium) was used, while the solution for the control plant did not contain any

chromium addition. The hydroponic solution's pH was adjusted to 6.0. the chromium-treated and

untreated control seedlings were cultured in a nutrient solution in a growth chamber (Temp. 260-280 ,

Humidity 70%-80%) with 10 hours of light and 14 hours of darkness (550–560 mmol s-1 per mA). The

nutritional solution was guaranteed to be continuously aerated. After seven days of culture, seedlings

treated with and without chromium were collected, and distinct roots and shoots were used for each

experiment.

Evaluation of morphological characteristics

Seven-day-old plant’s root length and shoot height were measured in centimeters. The roots and shoots

were then dried in an oven set to 80o C for two days in order to calculate their dry weight.

Measurement of electrolyte leakage

Electrolyte leakage (EL) was measured using the Lutts et al. (1996) methodology.

Determination of chlorophyll concentration:

A pre-chilled mortar and pestle was used to homogenize 100 mg of fresh leaf tissue with 5–10 mL of

90% (v/v) acetone and then centrifuged for five to ten minutes at 3000 rpm. To perform a

spectrophotometric analysis, the clear supernatant was collected. The concentration of chlorophyll in

leaves was measured with 90% (v/v) acetone based on the Lichtenthaler and Wellburn (1985) procedure.

Determination of Cr and Fe by AAS (atomic absorption spectroscopy)

Roots and shoots were first cleaned with CaSO4 and deionized water and then dried in an oven at 80o

C for three days. The samples were digested for 15 minutes at 100º C using mixes of HNO3 and H2O2

(3:1), and atomic absorption spectroscopy was used for investigation.

Determination of total soluble proteins

Fresh plant tissue was homogenized in 1-2 milliliters of ice-cold 50 mM phosphate buffer (pH 7.0). The

homogenate was centrifuged for 15 minutes at 4°C at 12,000 rpm and Quantification of proteins was

done using the supernatant. 100 µL of the sample and 1.0 mL of Bradford reagent were mixed and

allowed to sit at room temperature for 5 to 10 minutes. The absorbance was measured with a UV-Visible

spectrophotometer at 595 nm. Absorbance measurements were plotted against known BSA

concentrations to create a standard calibration curve. Each sample protein concentration was determined

by extrapolating the absorbance values from the standard curve.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 859 www.rsisinternational.org

Estimation of lipid peroxidation

The roots and shoots were homogenized with 5% (w/v) trichloroacetic acid (TCA) and centrifuged at

11,500×g for 15 minutes. Following centrifugation, thiobarbituric acid (TBA) was added to the

separated supernatant, then mixture was heated in a water bath for 30 minutes at 95 °C. As the mixture

cooled, absorbance was measured at 532 nm. Malondialdehyde was measured and reported as nmol of

MDA mg⁻¹ FW using an extinction value of 155 mM

⁻¹ cm

⁻¹ (Heath and Packer 1968).

Determination of rhizospheric pH change

A digital pH meter was employed to measure the media's pH both before and after the seedlings were

cultured. Using these data, the pH reduction was calculated and expressed as a percentage.

Silver nitrate precipitation test:

First, 0.1 M silver nitrate (AgNO₃) was prepared. Then a few drops of silver nitrate were added to each

tube holding the culture fluid. The organic acid indication, white precipitate, was seen.

Thin-layer chromatography (TLC)

Rice plant root exudates (BR-58, BR-63, BR-68, and BR-73), treated and untreated with Cr, were

analyzed using thin-layer chromatography (TLC). The mobile phase was made up of ethanol, NH4OH,

and H2O in the ratio 75.5:12.5:12.5. The TLC plate was then placed in the TLC chamber after the

placement of the samples and standards of known organic acids on it. The TLC plate is removed from

the chamber and allowed to dry when the solvent has travelled a sufficient distance. The TLC plate was

then examined under a UV light, and the spots were noted. The sample's organic acids were determined

by comparing them with known benchmarks.

Enzymatic analysis

The enzymes CAT (EC. 1.11.1.6), POD (EC. 1.11.1.7), SOD (EC. 1.15.1.1), and GR (EC. 1.6.4.2) were

extracted from one-week-old plants using a modified version of Goud and Kachole's (2012) approach.

After being crushed in 100 mM phosphate buffer, the roots and shoots tissues were centrifuged for 10

minutes at 13000×g. For the Catalase (EC. 1.11.1.6) analysis, the reaction mixture (2 ml) consisted of

400 µL of 6% (v/v) H2O2, 100 µL of root extract and 100 mM potassium phosphate buffer (pH 7.0). A

UV spectrophotometer was used to measure the absorbance at 240 nm (extinction coefficient of 0.036

mM-1 cm-1) at 30-second to one-minute intervals after the addition of root or shoot extract. The unit of

measurement for CAT activity is mmol of H2O2 oxidized min-1 (mg protein 1).

Similarly, two ml reaction mixture was prepared to measure peroxidase (EC 1.11.1.7) activity. Here 100

mM potassium phosphate buffer (pH 6.5), 1ml of 50 mM pyrogallol, 400 µL of 200 mM H₂O₂, and 100

µL of root extract as the enzyme source were all included in the combination. In the spectrophotometer,

the absorbance variations were measured from 30 seconds to 1.5 minutes at 430 nm (extinction

coefficient 12 mM-1 cm-1). The concentration of peroxidase is measured in mmol pyrogallol oxidized

min-1 (mg protein-1). Additionally, 50 mM sodium carbonate/bicarbonate buffer (pH 9.8), 0.1 mM

EDTA, 0.6 mM epinephrine, and enzyme made up the SOD (EC. 1.15.1.1) assay combination. Then

adrenochrome formation was observed at 475 nm using a UV-Vis spectrophotometer. The quantity of

enzyme required for 50% inhibition of epinephrine oxidation is established as per unit SOD activity. In

order to perform glutathione reductase (EC. 1.6.4.2) analysis, 100 µL of root extract was added to the

reaction mixture, which also contained 1 mL of 0.2 M phosphate buffer (pH 7.0), 1 mM EDTA, 0.75 ml

of distilled water, 0.1 mL of 20 mM oxidized glutathione (GSSG), and 0.1 mL of 2 mM NADPH. At

340 nm, the oxidation of NADPH by GR was then measured. The extinction coefficient of 6.12 mM-1

cm-1 was then used to calculate the rate of GR activity (nmol min-1).

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 860 www.rsisinternational.org

Determination of hydrogen peroxide and superoxide (O2-)

Tissues were centrifuged at 10,000×g for 15 minutes, after being pulverized in 0.1% (v/w)

trichloroacetic acid (TCA). Before reading the absorbance at 390 nm, the reaction mixture was made by

mixing potassium iodide (M) and phosphate buffer (10mM, pH7.0) with the supernatant. It was left in

the dark for an hour (Alexieva et al., 2001). A standard calibration curve with known H2O2 levels was

used for quantification.

For superoxide (O2-) measurement, plant samples were centrifuged at 5000 rpm for 7 minutes at 4 0C

after they had been crushed in 1 milliliter of 65 mM potassium phosphate buffer (pH 7.8). 50µl of 10

mM hydroxylamine hydrochloride, 0.5 ml of supernatant, and 450µl of 65 mM potassium phosphate

buffer (pH 7.8) were used to create the reaction mixture, which was then incubated for 30 minutes at 25

°C. After that, 125µl of 7mM alpha-napthyl amine and 10mM sulfanilamide were added to the mixture,

and then incubated for 20 minutes at 25 °C. The absorbance at 530 nm was measured using

spectrophotometry. Superoxide (O2-) levels were determined using a standard curve built with known

NO values.

Estimation of metabolites (Glutathione, phytochelatin and proline) content:

The method developed by Anderson et al. (1992) was used to extract glutathione. In order to determine

total glutathione, GSSG was first converted to GSH by mixing the leaf extract with 130 mM sodium

phosphate buffer (pH 7.4) and one unit of glutathione reductase. The mixture was then kept at 30°C for

10 minutes. The final reaction mixture was then made by adding 50 mM of NADPH and 7 mM of

sodium phosphate buffer (pH 6.8) that contained 6 mM of DTNB. Absorbance was measured at 412 nm

after this reaction mixture was held at 30°C for 10 minutes. To estimate glutathione, a standard curve of

known quantities of GSH was employed (Griffith 1980). Phytochelatin content was determined using

the previously described procedure by Mahmud et al. (2018).

To measure the amount of proline samples of leaves and roots were centrifuged at 11,500×g for 12

minutes after being homogenized in 3% (v/w) sulfosalicylic acid. Next, the 100 μL plant extract

supernatant was mixed with 200 μL glacial acetic acid, 100 μL of 3% (v/w) sulfosalicylic acid, and 200

μL acidic ninhydrin. The mixture was then heated for 60 minutes at 96◦C and immediately chilled on

ice. Spectrophotometer was used to take a reading at 520 nm (Bates et al., 1973). Calculations were

performed using a standard curve with known proline concentrations.

Statistical analysis

Completely randomized block design with four independent replications was adopted in each

experiment.t-test at 0.05% significance level with the help of Microsoft Excel 2007 was performed as

statistical analysis. Moreover, Graph Pad Prism was applied to prepare graphical presentations.

RESULT

Morpho-physiological parameters:

Seedlings grown on the hydroponic solution containing Cr showed retardation of growth in most of the

rice varieties except BR-58, BR-63, BRRI-68, and BR-73. No significant shoot and root length as well

as root and shoot dry weight reduction were observed in these four varieties for chromium toxicity

compared with the control (table. 1). Moreover, chlorophyll concentrations in the shoot of these four

varieties remained unchanged under chromium stress, whereas in other varieties, it decreased

meaningfully under chromium stress.

Furthermore, no meaningful differences between control and chromium treated plant was observed in

these four varieties (BR-58, BR-63, BRRI-68, and BR-73) in case of electrolyte leakage. But in

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 861 www.rsisinternational.org

remaining varieties electrolyte leakage was found to be increased significantly under chromium stress

equated to control. Considering the above-mentioned parameters BRRI-58, BRRI-63, BRRI-68 and

BRRI-73 found to cope with chromium toxicity.

Table 1. Morpho-physiological features of 25 high yielding rice varieties grown in absence or presence

of Chromium on hydroponic solution. Different letters indicate significance difference between means

of treatments (number of replications is 4) followed by t-test. Data were from one-week plats.

Variety Treatment

Root

length

(cm)

Root dry

(mg)

Shoot

length

(cm)

Shoot dry

(mg)

Total

Chlorophyll

(µgm/mg)

Electrolyte

leakage in

root (µs cm

-1

)

BRRI-

Control 4.83±0.29

2.67±0.58

7.5±.5

7.33±0.58

130.58±5.56

0.955±0.08

Treatment 3.5±0.5

0.77±0.25

4.83±0.29

5.33±0.58

79.27±5.77

1.750±0.23

BRRI-

Control 6±0.87

2±0

10.5±0.87

5.33±0.58

255.18±47.22

0.915±0.60

Treatment 2.17±0.58

0.36±0.13

3.17±0.76

2.33±0.58

162.30±20.14

1.962±0.86

BRRI-

Control 7.83±1.44

2±0

8.5±0.5

6.83±0.29

290.57±8.92

1.263±0.15

Treatment 3.67±1.15

0.83±0.29

7.17±0.29

5.5±0.5

268.09±20.79

2.568±0.81

BRRI-

Control 9.67±0.58

4.33±0.58

11.33±0.58

12.67±0.58

200.24±11.85

0.531±0.41

Treatment 5.83±0.29

1.77±0.87

8.17±0.76

10.67±0.58

180.93±3.28

1.502±0.58

BRRI-

Control 8±1

3.33±0.58

11±0.5

9.33±0.58

309.21±3.32

0.789±0.18

Treatment 6.17±0.29

2±0

9±0.5

7.67±0.58

288.94±3.17

1.669±0.59

BRRI-

Control 5±0.87

3.83±0.29

9±0.5

8.67±1.15

424.33±42.49

0.869±0.29

Treatment 3.5±0

3±0

9±0

8.83±1.04

220.44±78.13

1.718±0.307

BRRI-

Control 7.33±0.29

3.83±0.29

13.83±0.76

10.17±0.29

227.89±31.12

0.955±0.21

Treatment 6.33±0.58

2.67±0.29

11.33±0.58

8.66±0.58

216.90±20.56

1.483±0.48

BRRI-

Control 6.67±0.58

4±0.5

10.67±0.58

8.67±0.29

416.20±170.96

1.203±0.08

Treatment 5.33±0.29

3±0

9.33±0.58

7.33±0.29

387.21±48.55

1.758±0.19

BRRI-

Control 8.33±0.58

1.83±0.29

9.83±0.58

7.83±0.29

264.99±44.99

1.083±0.14

Treatment 6.5±0.5

0.93±0.12

8±0.87

6.67±0.58

232.14±58.09

2.145±0.74

BRRI-

Control 9.17±2.02

3.33±0.58

10.83±0.76

8.5±0.5

262.39±78.84

1.735±0.23

Treatment 7.33±2.31

2.67±1.15

10.5±0.5

8.33±0.58

300.94±42.66

1.902±0.81

BRRI-

Control 8.17±0.76

2.83±0.29

8±1

8.67±0.29

454.60±54.07

1.259±0.35

Treatment 5.67±0.58

2±0

6±1

7.5±0.5

211.31±52.26

2.185±0.16

BRRI-

Control 9±0

2.33±0.58

13.83±0.29

10.33±0.58

347.63±8.61

1.865±0.56

Treatment 7.67±0.58

1.67±0.58

11.5±0.87

9.17±1.15

316.30±40.03

2.026±0.46

BRRI-

Control 6.67±0.58

3.67±0.58

9±0

8±0

274.89±6.00

0.997±0.58

Treatment 4±1.73

2.33±0.58

7.67±0.58

6.67±0.58

165.69±57.69

1.941±0.49

BR-62 Control 6.33±2.25

2.67±0.58

9±2.783

6.67±2.52

363.50±40.25

0.819±0.18

Treatment 6.33±3.21

3±1

9.5±2.179

6.66±2.08

282.39±38.00

0.925±0.44

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 862 www.rsisinternational.org

BRRI-

Control 3.33±0.28

3.33±0.58

10.83±0.58

8±1

310.99±88.42

1.932±0.913

Treatment 4.5±0.87

2.67±0.58

10.17±1.89

7.33±0.58

306.85±26.59

1.944±0.48

BRRI-

Control 9.33±0.58

4±0

10.67±0.76

10.33±0.58

465.75±10.17

1±0

Treatment 6.67±0.58

3.17±0.29

9.33±0.29

9.17±0.29

382.09±15.69

1.825±0.72

BRRI-

Control 3.33±1.15

4.17±1.26

11.5±1.80

10±0

336.40±30.52

1.189±0.27

Treatment 5.5±1.80

3.67±0.58

13.67±1.61

9.33±0.58

173.67±44.59

1.653±0.74

BRRI-

Control 8.33±1.53

3.67±0.58

9±1

7±0

414.54±38.93

0.766±0.12

Treatment 4.67±0.58

2.5±0.5

7.17±0.29

5.67±0.58

263.36±20.61

1.516±0.41

BRRI-

Control 5.67±0.58

4±1.73

11±0.5

9.33±0.58

512.35±43.78

0.976±0.24

Treatment 4.13±0.55

0.93±0.12

9.17±0.29

8.33±0.29

209.97±89.81

2.222±0.48

BRRI-

Control 4.33±1.53

2.33±0.58

7.17±2.84

9.67±1.53

251.98±22.95

0.869±0.10

Treatment 4.83±1.89

2.83±0.29

7.5±3.04

9.33±0.58

268.57±76.08

0.942±0.64

BRRI-

Control 5.17±1.04

3.33±0.58

9.5±0

7.33±0.58

430.47±53.04

1.136±0.460

Treatment 6.17±2.57

4.33±3.21

8.83±2.08

7.33±2.08

262.21±42.71

1.031±0.540

BRRI-

Control 4.33±2.57

1.67±0.29

10.67±1.15

6.83±0.58

829.50±93.83

1.018±0.57

Treatment 3±0

0.87±0.12

6.5±0.5

4.33±0.58

424.15±79.69

1.361±0.591

BRRI-

Control 7.5±0.5

3.17±0.76

10±0.87

9.33±0.58

459.82±48.06

0.892±0.11

Treatment 6.17±0.29

2.17±0.29

7±0.87

7.5±0.5

326.48±75.24

1.761±0.540

BRRI-

Control 11.33±0.58

3.5±0.5

9.17±0.76

9±1

393.12±112.24

1.253 ±0.60

Treatment 7.33±1.15

2.17±0.29

7.33±0.29

5.33±0.58

375.90±68.46

2.142±0.618

BRRI-

Control 8.33±0.29

3.5±0.5

9.33±1.53

8±1

352.32±87.64

0.989±0.23

Treatment 7±0

2.17±0.29

11±1

9±1

232.54±28.91

1.538±0.3

Chromium and Iron content:

As the initial investigation indicates that BR-58, BR-63, BRRI-68, and BR-73 varieties owned the

detoxification mechanism, analysis of chromium concentration in root and shoot was conducted in these

four varieties. Root's chromium content of rice varieties BR-58, BR-63, and BR-68 was significantly

higher under chromium stress compared with the control plant. But in the shoot, no meaningful

differences in chromium content between control and chromium-stressed plants of the varieties were

observed (Fig. 1). This analysis concludes that chromium translocation from root to shoot was inhibited

in varieties BR-58, BR-63, and BR-68 under the stressed condition.

Furthermore, significant iron content compared with the control plant was found only in the root of

varieties BR-58 and BR-63 and in the shoot of BR-58 among the varieties grown under chromium stress.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 863 www.rsisinternational.org

(

)

(

)

(

)

(

)

(

)

(

)

(

)

(

)

200

400

600

Iron content (µg/gm DW)

Control

Chromium

Figure 1. Chromium (A) and Iron (B) concentrations in rice seedlings grown under chromium stress

and non-stress condition. Significant deviations among the treatments were denoted by different letters

followed by t-test at 5% (P<0.05) significance level (number of replications is 4)

Lipid peroxidation and total soluble protein:

Malondialdehyde (MDA) content both in root and shoot of varieties BR-58, BR-63, BR-68 and BR-73

showed no significant differences between non treated control and treatments indicating the tolerance

of these varieties against chromium induced oxidative damage (Fig. 2).

Moreover, no significant difference of total soluble protein content between control and chromium

stressed plants except in the root of variety BR-63.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 864 www.rsisinternational.org

Figure 2: Malondialdehyde (A) and total soluble protein content (B) in the roots and shoots of 7 days

old rice plants (BR-58,63,68,73) grown under Chromium stress and without chromium stress. Different

letters in each column indicate significant differences between means of treatments followed by t-test at

5% significance level (number of replications is 4).

Determination of root secreted organic acids and rhizospherep Hreduction rate:

Rhizosphere pH decrease after culture (due to low molecular weight organic acids released by rice roots)

on the Hongland solution was mentioned in all rice varieties, including the chromium-sensitive variety

BR-51, in which it was less than 10% in both stress and non-stress conditions (fig. 3). But in BR-58,

BR-63, BR-68 and BR-73 (treated as chromium tolerant) this reduction rate was over 15% under

chromium stress conditions. The required amount of low molecular weight organic acids (LMWOA)

release is assured by more than 15% rhizospheric pH reduction to avoid chromium toxicity.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 865 www.rsisinternational.org

Furthermore, rhizospheres with less than 10% pH reduction could not release a sufficient amount of

LMWOA to cope with chromium toxicity.

However, a white precipitate in the silver nitrate test supports that the root of rice seedlings releases

organic acid. Furthermore, thin-layer chromatographic analysis confirmed that both the Cr-treated and

non-treated rice seedlings secreted citric acid.

Figure-3: A) Rhizosphoric pH reduction rate (%) under Chromium stress and without chromium stress.

B) TLC paper with spots of organic acid C) precipitation of organic acids in AgNO₃ test

Enzymatic activity:

CAT and POD activities in the roots of the varieties BR-58, BR-63 and BR-68 grown under chromium

stress were significantly increased when compared with the control plant (Table. 2). GR activities in the

roots of varieties BR-58 and BR-68 were also increased due to chromium. Moreover, enhancement of

SOD activities was observed only in the root of variety BR-68 for toxic chromium. Moreover, no

mentionable changes of enzymatic activities (CAT, POD, SOD and GR) were observed in roots between

control and treated plants of variety BR-73.

Furthermore, no mentionable enhancement of CAT, POD, and SOD activities was followed in the shoots

of varieties BR-58, BR-63, BR-68, and BR-73 under chromium stress. But GR activity in the shoot of

varieties BR-58, BR-68 and BR-73 grown under chromium stress was found to be increased

significantly compared with the control plant.

Furthermore, reactive oxygen species (ROS) especially superoxide (O

) and hydrogen peroxide (H

)

were also estimated. H

content in the root of varieties BR-58, 63 and 68 was enhanced meaningfully

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 866 www.rsisinternational.org

than that of control plant but in the shoot of these varieties, no meaningful changes were observed. One

the other hand no significant differences of superoxide (O

) concentration was discovered except in the

root of BR-68.

Table 2 : Changes in enzyme activity in roots and shoots of rice varieties grown in the presence or

absence of chromium. Different letters indicate significant differences between mean (including

standard deviation) of treatments (number of replication is 4) followed by t-test. Data were from one

week old plants.

BR-58 BR-63 BR-68 BR-73

CAT (min-1protein-

Cr-

4.27±1.01

4.29±1.37

1.02±0.87

0.64±0.53

Cr+

7.08±0.51

7.80±0.35

2.34±0.38

0.62±0.48

POD (min-1protein-

Cr-

4.01±0.02

1.36±0.79

1.45±0.22

0.94±0.57

Cr+

7.90±0.75

3.80±0.54

2.51±0.44

0.64±0.35

SOD (min-1protein-

Cr-

8.0x10

-2

±1.0x10

-2

6.0x10

-2

±7.0x10

-2

1.0x10

-2

±1.0x10

-2a

4.0x10

-2

±1.0x10

-2a

Cr+

8.0x10

-2

±0

3.0x10

-2

±1.0x10

-2a

5.0x10

-2

±1.0x10

-2b

2.0x10

-2

±0

GR (nmol.NADH.

min-1gm protein-1)

Cr-

2.0x10

-2

±1.0x10

-2a

8.0x10

-2

±2.0x10

-2a

4.0x10

-2

±0

5.0x10

-2

±2.0x10

-2

Cr+

3.0x10

-2

±1.0x10

-2b

6.0x10

-2

±1.0x10

-2a

5.0x10

-2

±1.0x10

-2b

5.0x10

-2

±1.0x10

-2a

Super Oxide(O2-)

(µg/mg)

Cr-

0.526±o.24

1.011±0.32

0.706±0.12

0.528±0.09

Cr+

0.606±0.16

1.469±0.32

0.476±0.03

0.569±0.08

H2O2 (µg/mg) Cr-

1.2x10

-2

±0.3x10

-2a

2.7x10

-2

±1.0x10

-2a

2.9x10

-2

±1.0x10

-2a

8.1x10

-2

±1.0x10

-2a

Cr+

4.3x10

-2

±0.8x10

-2b

79.8x10

-2

±3.0x10

-2b

7.4x10

-2

±1.0x10

-2b

10.2x10

-2

±2.0x10

-2a

CAT (min-1protein-

Cr-

0.55±0.27

2.66±0.39

0.88±0.68

0.56±0.34

Cr+

0.79±0.31

2.81±1.98

0.95±0.05

0.82±0.19

POD (min-1protein-

Cr-

1.31±0.18

1.04±0.16

1.86±0.21

0.46±0.06

Cr+

1.79±0.38

1.59±0.35

2.02±0.12

0.61±0.09

SOD (min-1protein-

Cr-

2.0x10

-2

±1.0x10

-2a

2.0x10

-2

±0

2.0x10

-2

±1.0x10

-2a

2.0x10

-2

±1.0x10

-2a

Cr+

3.0x10

-2

±0

2.0x10

-2

±1.0x10

-2a

2.0x10

-2

±0

3.0x10

-2

±0

GR (nmol.NADH.

min-1gm protein-1)

Cr-

2.0x10

-2

±0

2.0x10

-2

±1.0x10

-2a

2.0x10

-2

±0

2.0x10

-2

±0

Cr+

3.0x10

-2

±0

2.0x10

-2

±1.0x10

-2a

3.0x10

-2

±0

3.0x10

-2

±1.0x10

-2b

Super Oxide(O2-)

(µg/mg)

Cr-

0.542±0.08

0.810±0.19

0.152±0.04

0.271±0.15

Cr+

0.579±0.17

0.585±0.09

0.237±0.04

0.342±0.14

H2O2 (µg/mg) Cr-

2.7±2.0x10

-2a

1.5x10

-2

±1.0x10

-2a

1.5x10

-2

±1.0x10

-2a

2.4x10

-2

±1.0x10

-2a

Cr+

2.3x10

-2

±2.0x10

-2

2.0x10

-2

±1.0x10

-2a

1.4x10

-2

±1.0x10

-2a

2.9±1.0x10

-2a

Metabolites (glutathione, proline and phytochelatin)

Amount of glutathione in the root of variety BR-58 and BR-68 as well as in shoot of varieties BR-58,

BR-68 and BR-73 were significantly raised under chromium stress compared with non -treated control

plant (Table 3). However, no significant change of glutathione content was followed both in root and

shoot of variety BR-63.

Moreover, secondary metabolite proline was significantly increased in roots of varieties BR-58, BR-63

and BR-68 under chromium stress compared with control plants. But a meaningful difference of proline

content in the shoot of these varieties was not observed.

Furthermore, phytochelatin content in the root of BR-58 was significantly increased when grown on

hydroponic solution supplemented with chromium compared with non-treated control plants. But in the

shoot of all varieties, it was not increased significantly.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 867 www.rsisinternational.org

Table 3: Metabolites in roots and shoots of rice varieties grown in the presence or absence of chromium.

Different letters indicate significant differences between mean (including standard deviation) of

treatments followed by t-test (number of replications is 4). Data were from one week old plants.

Glutathione

(µgm/mg FW)

Proline

(µgm/mg FW)

Phytochelatine

(µgm/mg FW)

BR-58 Cr- 1.6x10-2±0.1x10 -2a 12.4x10-2±1.2x10 3.4x10-2±0.7x10 -2a

Cr+ 2.0x10-2±0.7x10 -3b 18.2x10-2±2.8x10 -2b 4.9x10-2±0.7x10 -2b

BR-63 Cr- 1.8x10-2±0.4x10 -2a 14.7x10-2±1.7x10 -2a 5.2x10-2±1.5x10 -2a

Cr+ 1.9x10-2±0.3x10 -2a 24.9x10-2±1.8x10 -2b 6.7x10-2±2.1x10 -2a

Br-68 Cr- 1.2x10 2±0.1x10 -2a 8.2x10-2±2.1x10 -2a 3.7x10-2±0.4x10 -2a

Cr+ 1.8x10-2±0.3x10 -3b 1.1x10-2±2.2x10 -2b 4.2x10-2±1.2x10 -2a

BR-73 Cr- 1.6x10-2±0.2x10 -2a 11.3x10-2±1.3x10 -2a 6.8x10-2±0.3x10 -2a

Cr+ 1.6x10-2±0.2x10 -2a 15.0x10-2±5.2x10 -2a 6.9x10-2±0.7x10 -2a

BR-58 Cr- 1.3x10-2±0.46x10 -2a 15.1x10-2±6.1x10 -2a 4.9x10-2±0.2x10 -2a

Cr+ 2.8x10-2±0.3x10 -2b 17.9x10-2±3.9x10 -2a 4.9x10-2±0.8x10 -2a

BR-63 Cr- 1.7x10-2±0.4x10 -2a 32.2x10-2±8.6x10 -2a 4.6x10-2±0.5x10 -2a

Cr+ 1.7x10-2±0.4x10 -2a 31.1x10-2±6.9x10 -2a 5.2x10-2±0.9x10 -2a

Br-68 Cr- 1.5x10-2±0.2x10 -3a 16.5x10-2±6.5x10 -2a 4.1x10-2±0.5x10 -3a

Cr+ 1.7x10-2±0.6x10 -3b 14.6x10-2±2.9x10 -22a 4.9x10-2±0.7x10 -2a

BR-73 Cr- 1.9x10-2±0.2x10 -2a 12.4x10-2±2.8x10 -2a 7.1x10-2±0.6x10 -2a

Cr+ 2.4x10-2±0.2x10 2b 9.7x10-2±1.4x10 -2a 7.0x10-2±1.4x10 -2a

DISCUSSION

Heavy metal stress significantly affects the physiology of plants (Rehman, et, al., 2021, Rolf et.al, 2004)

and crop production is reduced as a consequence (Jewell et al., 2010). On the other hand, plants also

cope with heavy metal toxicity through the mechanism of avoidance and tolerance (Yu et, al., 2019).

Metal-tolerant rice varieties are proving to be a boon for the farmers. Plant breeders employ selection

method on a large number of plants cultivated under metal stress to assess heavy metal-tolerant varieties.

However, assessment of tolerance level is very crucial for selecting a tolerant variety as it varies among

the different species or varieties. In this investigation, twenty-five authentic high-yielding rice varieties

were developed in the chromium treated hydroponic solution as treatment and without chromium as

control. The result of chromium toxicity on different morphological and physiological parameters such

as root and shoot length, total chlorophyll content, and electrolyte leakage in rice plants was identified

by comparing with control plants, as these parameters are significantly hampered in rice plant for

chromium (Riaz et, al., 2024, Khatun, et. al, 2019). Fargasova (2001) also mentioned that photosynthetic

pigments as well as photosynthetic processes are damaged for toxic metals. However, in this study,

varieties BR-58, BR-63, BR-68, and BR-73 were proved chromium tolerant based on morpho-

physiological features. Moreover, constant level of total protein and MDA content under chromium

stress compared with control plants, indicates resistance against chromium as higher lipid peroxidation,

cut down protein level, is well documented in rice against chromium toxicity (Khatun, et. al., 2019,

Mukta, et.al, 2019)

Analysis of chromium content in root and shoot exhibits that chromium translocation to shoot is

inhibited compared with the control plant in variety BR-58, BR-63 and BR-68. Vacuolar sequestration

can restrict heavy metal chromium in root cells vacuoles (Huda et al., 2017) with the help of thiol (SH)-

containing molecule Phytochelatin and helps plants to survive under stress (Huda et al., 2017). In the

present investigation, in variety BR-58 and BR-68, a significant increase of phytochelatin content and

its precursor glutathione equated with control plants ensure the chromium sequestration in roots.

However, in variety BR-63 no enhanced phyochelatine ensure that low molecular weight citric acid play

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 868 www.rsisinternational.org

vital role on vacuolar sequestration. Because citrate function as counterions being stored in the plant

cell vacuole (Martinoia, et.al,1994 and Meyer et.al., 2011). A similar result was also found in rice under

Cd stress due to exogenous silicon application (Bari, et.al, 2020).

Plant roots absorb metal ions stored in the xylem, form a complex with the chelator, and then transport

them to the shoot. The molecules that function as chelators inside the cell to sequester the heavy metals

in the cell vacuole are organic acids, amino acids, and phosphate derivatives (Rauser 1999). In rice,

exogenous application of organic acids enhances heavy metal uptake and transport to the aerial portion

through the xylem (Khatun et. al., 2019) as well as sequesters in cell vacuoles (Huda et. al., 2016). On

the other hand, secreted organic acids by plant roots form non-toxic compounds with heavy metals by

their carboxyl groups and prevent their entrance to the plant (Guan, et. al., 2024, Yu G et al 2019).

Previous investigations indicate that plants release significant amounts of low molecular organic acids

(LMWOAs) in response to heavy metal toxicity, which is an exclusion mechanism rendering metal

uptake and also unique to each species (Guan, et, al., 2024, Montiel-Rozas et. al., 2016). Lowering pH

significantly influenced plant metal uptake that enhance phytoextraction strategy (Wang, et. al., 2006).

However, the release of organic acid was well documented by a white precipitate in the silver nitrate

test in this study. Moreover, thin-layer chromatographic analysis confirmed that both the Cr-treated and

non-treated rice seedlings secrete citric acid. In this investigation, reduction of rhizospheric pH (more

than 15%), as well as decreased translocation of Cr to shoots in varieties BR-58, BR-63 and BR-68

indicates secreted LMWOAs confer metal tolerance by sequester mechanism. Moreover, in variety BR-

73, reduced rhizospheric pH (more than 15%) and reduced chromium uptake propose an effective

exclusion mechanism. Zeng Fanrong et.al., (2008) reported that rice plants released oxalic, malic, and

citric acid at the rhizosphere and enhanced Cr accumulation. But this report could not provide any

evidence regarding the amount of low molecular organic acids (LMWOAs) secretion to confer

chromium tolerance as well as any other mechanism of chromium detoxification rather than

accumulation, as the investigation was limited to two rice genotypes.

However, stability constant of OA-metal complexes specifies the detoxification capacity of organic

acids. Chelating complex between Al and citrate in carrot form at the ratio 1:1 where oxalate form

complex with Al at the ratio of 1:3 (Kyoma et.al., 1990) indicating that stability constant is variable

among different ratio. These two organic acids ensure detoxification mechanism by preventing Al to

bind ATP or other ligands. Moreover, in aquatic solution, higher concentration of malate is required to

alleviate Al rhizotoxicity through chelation (Thomas et.al., 2005). Aluminum-OA complex is

precipitated in apoplast and excrete more malate in Al-tolerant wheat than the Al-sensitive wheat variety

(Ryan et.al., 1995).

Furthermore, Consumption of carbon sources for production and efflux of Organic acids (OA) under

stress consumed significant proportion of carbon imposing an energy cost to plants which is

economically important for fast-growing annual crops like rice (Koyama et al., 2000; Herz et al., 2018).

Furthermore, plants optimize its carbon loss by limiting the amount of OAs release by negative

regulators of OA exudation like GABA (Ramesh et al., 2015).

But in our investigation, it was evident that the required amount of citric acid secretion to cope with

chromium toxicity was assured by more than 15% rhizospheric pH reduction, whereas sensitive rice

plants reduce less than 10% rhizospheric pH.

It is the first report that provides rice varieties (BR-58, 63, 68 and 73) ignore its carbon optimization

process to produce citric acids for coping with heavy metal chromium. Moreover, rice plant release citric

acid to such a height that can neutralize chromium and it measured by its 15% rhizospheric pH

reduction. Our investigation also concludes that 15% and above rhizospheric pH reduction in rice plant

is the benchmark for required amount of citric acid secretion to be chromium tolerant. This investigation

also discovered that under chromium stress, root secreted LMWOAs adopted two mechanisms such as

vacuolar sequestration through chelation, and metal exclusion to avoid chromium toxicity in rice

varieties

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 869 www.rsisinternational.org

However, limited activity of Fe transporters can reduce Fe uptake, which can enhance the tolerance level

of rice plants against Cr toxicity (Kabir 2016). But in the present study, Fe concentration in the root and

shoot of these four varieties was not followed when compared with the control plant, indicating that

regulation of Fe transporter was not involved with chromium tolerance.

Oxidative stress generated from ROS is pronounced in the plant cells during metal toxicity, and

activation of antioxidant mechanisms is initiated (Ghori, 2019). Reduce protein content, lipids

peroxidation, inefficient enzyme activities, DNA damage and abnormal constituents of cells are the

result of excessive accumulations of reactive oxygen species (ROS) in plant. ROS also interacts with

hormones and epigenetic modifiers to regulate developmental processes and stress responses of plant

(Kong et al., 2018). In this present investigation, under chromium stress, ROS was found to be increased

in the root of these varieties without any significant lipid peroxidation compared with the control plant.

Two types of mechanisms are present in the plant to scavenge the ROS, and the first one is enzymatic

and the other one is non-enzymatic. Hydrogen peroxide (H2O2) generated first from oxidized metabolic

and then convert into water by means of enzymatic activities while termination of free radical chain

reactions by the help of non-enzymatic antioxidants (Moussa Ziad 2019). Neutralization of ROS by

through enzymatic process enhance the plant tolerance level against diverse biotic and abiotic stresses

(Kanto et al. 2015). In the present study, GR activity was enhanced significantly in the root of varieties

BR-58 and BR-68 and the shoot of varieties BR-58, BR-68, and BR-73 under chromium stress.

Furthermore, , SOD activity was found to be increased only in the root of variety BR-68 under same

stress. Enhanced GR and SOD activity reported in rice under Cd stress also (Bari, et.al., 2020)

Glutathione, a non-enzymatic antioxidant, is an efficient scavenger of O2, H2O2 and OH (Gill and

Tuteja, 2010). In our study, the amount of glutathione in the roots of varieties BR-58 and BR-68 as well

as in the shoots of varieties BR-58, BR-68 and BR-73 was significantly raised under chromium stress.

However, our investigation again notices that citric acid up regulate the antioxidant defense system as a

secondary function along with chromium avoidance mechanisms to keep down ROS levels.

Proline (Pro) an amino, responsible to mitigate biotic and abiotic stress in plant (Ágneset.al., 2018). It

is also reported as metals chelator (Gill and Tuteja, 2010). In the present investigation, proline content

was found to be enhanced significantly in the roots of BR-58, BR-63, and BR-68 to enhance the

tolerance against Cr toxicity. The findings of this study hold practical significance for both crop improvement

and environmental remediation.

CONCLUSION

The exudation of citric acid has been shown to be the key physiological response against chromium

toxicity in rice plant. But the concentration of citric acid is deviated according to the genotypes of rice

plants. The assessment of the amount of citric acid exudates followed by rhizosphere pH reduction rate

has allowed knowing the potentiality of rice varieties to cope with chromium toxicity. Furthermore, rice

genotypes that can reduce their rhizosphere pH by 15% or more by releasing organic acid are chromium

tolerant. However, the findings offer an efficient screening technique for metal tolerant rice plants.

DECLARATION

Funding: This research did not receive any specific grant from funding agencies in public, commercial

or not for profit sectors.

Conflicts of interest/Competing interests: The authors declare that they have no conflict of interest.

Ethics approval: Not applicable

Consent to participate: All participants are in the list of authors and have full consent

Consent for publication: All participants are in the list of authors and have full consent

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 870 www.rsisinternational.org

Availability of data and material: The datasets are available from the corresponding author on

reasonable request.

Generative AI: Not applicable

ACKNOWLEDGEMENTS

I am grateful to Bangladesh Rice Research Institute (BRRI) for providing seeds of rice varieties. "The

germplasm used in this study was obtained from the Bangladesh Rice Research Institute (BRRI) through

their official procedure, which involved submitting a formal application stating the purpose of use. The

institute evaluated and approved the request prior to providing the seeds. No separate permission letter

was issued after seed distribution, as approval was granted at the initial stage

REFERENCE

1. Ágnes Szepesi,Réka Szőllősi (2018). Mechanism of Proline Biosynthesis and Role of Proline

Metabolism Enzymes Under Environmental Stress in Plants, in:Parvaiz Ahmad, Mohammad

AbassAhanger, Mohammed Nasser Alyemeni (Eds), Plant Metabolites and Regulation Under

Environmental Stress. Academic press.pp 337-353.

https://doi.org/10.1016/B978-0-12-812689-

9.00017-0

2. Airaki M, Leterrier M, Mateos RM, Valderrama R, Chaki M, Barroso JB, Del Río LA, Palma

JM, Corpas FJ. (2012). Metabolism of reactive oxygen species and reactive nitrogen species in

pepper (Capsicum annuum L.) plants under low temperature stress. Plant, Cell & Environment

35: 281–29

3. Al Mahmud, J., Hasanuzzaman, M., Nahar, K., Bhuyan, M. B., & Fujita, M. (2018). Insights

into citric acid-induced cadmium tolerance and phytoremediation in Brassica juncea L.:

Coordinated functions of metal chelation, antioxidant defense and glyoxalase systems.

Ecotoxicology and environmental safety, 147, 990-

1001.https://doi.org/10.1016/j.ecoenv.2017.09.045.

4. Alexieva, V., Sergiev, I., Mapelli, S., & Karanov, E. (2001). The effect of drought and ultraviolet

radiation on growth and stress markers in pea and wheat. Plant, Cell & Environment, 24(12),

1337-1344.https://doi.org/10.1046/j.1365-3040.2001.00778.x.

5. Al-Khayri, J. M., Rashmi, R., Toppo, V., Chole, P. B., Banadka, A., Sudheer, W. N., ... & Rezk,

A. A. S. (2023). Plant secondary metabolites: The weapons for biotic stress

management. Metabolites, 13(6), 716. https://doi.org/10.3390/metabo13060716.

6. Anderson, J. S., Lall, S. P., Anderson, D. M., &Chandrasoma, J. (1992). Apparent and true

availability of amino acids from common feed ingredients for Atlantic salmon (Salmo salar)

reared in sea water. Aquaculture, 108(1-2), 111-124.https://doi.org/10.1016/0044-

8486(92)90322-C.

7. Bari, M. A., Prity, S. A., Das, U., Akther, M. S., Sajib, S. A., Reza, M. A., & Kabir, A. H. (2020).

Silicon induces phytochelatin and ROS scavengers facilitating cadmium detoxification in

rice. Plant Biology, 22(3), 472-479.https://doi.org/10.1111/plb.13090.

8. Bates, L. S., Waldren, R. P. A., & Teare, I. D. (1973). Rapid determination of free proline for

water-stress studies. Plant and soil, 39, 205-207.https://doi.org/10.1007/BF00018060.

9. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Analytical

biochemistry, 72(1-2), 248-254. https://doi.org/10.1016/0003-2697(76)90527-3.

10. Dey, S. R., Sharma, M., & Kumar, P. (2023). Effects and responses of chromium on plants.

In Chromium in Plants and Environment (pp. 385-427). Cham: Springer Nature Switzerland.

https://doi.org/10.1007/978-3-031-44029-8_14.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 871 www.rsisinternational.org

11. Dong D, Peng X, Yan X. (2004). Organic acid exudation induced by phosphorus deficiency

and/or aluminium toxicity in two contrasting soybean genotypes. Physiologia Plantarum 122:

190–199

12. Fargašová, A. (2001). Phytotoxic effects of Cd, Zn, Pb, Cu and Fe on Sinapis alba L. seedlings

and their accumulation in roots and shoots. Biologia plantarum, 44, 471-473.

https://doi.org/10.1023/A:1012456507827.

13. Fernando DR, Mizuno T, Woodrow IE, Baker AJ, Collins RN (2010). Characterization of foliar

manganese (Mn) in Mn (hyper)accumulators using X-ray absorption spectroscopy. New

Phytologist 188:1014–1027.

14. Ghori, N. H., Ghori, T., Hayat, M. Q., Imadi, S. R., Gul, A., Altay, V., & Ozturk, M. (2019).

Heavy metal stress and responses in plants. International journal of environmental science and

technology, 16, 1807-1828. https://doi.org/10.1007/s13762-019-02215-8.

15. Gill, S. S., & Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic

stress tolerance in crop plants. Plant physiology and biochemistry, 48(12), 909-930.

http://dx.doi.org/10.1016/j.plaphy.2010.08.016.

16. Goud, P. B., &Kachole, M. S. (2012). Antioxidant enzyme changes in neem, pigeonpea and

mulberry leaves in two stages of maturity. Plant signaling & behavior, 7(10), 1258-1262.

https://doi.org/10.4161/psb.21584.

17. Griffith, O. W. (1980). Determination of glutathione and glutathione disulfide using glutathione

reductase and 2-vinylpyridine. Analytical biochemistry, 106(1), 207-212.

https://doi.org/10.1016/0003-2697(80)90139-6.

18. Guan, J., Zhang, Y., Li, D., Shan, Q., Hu, Z., Chai, T., ... & Qiao, K. (2024). Synergistic role of

phenylpropanoid biosynthesis and citrate cycle pathways in heavy metal detoxification through

secretion of organic acids. Journal of Hazardous Materials, 476, 135106.

https://doi.org/10.1016/j.jhazmat.2024.135106

19. Harada, E., Kim, J. A., Meyer, A. J., Hell, R., Clemens, S., & Choi, Y. E. (2010). Expression

profiling of tobacco leaf trichomes identifies genes for biotic and abiotic stresses. Plant and cell

physiology, 51(10), 1627-1637.https://doi.org/10.1093/pcp/pcq118

20. Heath, R. L., & Packer, L. (1968). Photoperoxidation in isolated chloroplasts: I. Kinetics and

stoichiometry of fatty acid peroxidation. Archives of biochemistry and biophysics, 125(1), 189-

198.https://doi.org/10.1016/0003-9861(68)90654-1.

21. Herz K, Dietz S, Gorzolka K, Haider S, Jandt U, Scheel D, Bruelheide H. (2018). Linking root

exudates to functional plant traits. PLoS One 13: e0204128.

22. Huda, A. N., Haque, M. A., Zaman, R., Swaraz, A. M., & Kabir, A. H. (2017). Silicon

ameliorates chromium toxicity through phytochelatin-mediated vacuolar sequestration in the

roots of Oryza sativa (L.). International journal of phytoremediation, 19(3), 246-

253.https://doi.org/10.1080/15226514.2016.1211986.

23. Huda, A. N., Swaraz, A. M., Reza, M. A., Haque, M. A., & Kabir, A. H. (2016). Remediation

of chromium toxicity through exogenous salicylic acid in rice (Oryza sativa L.). Water, Air, &

Soil Pollution, 227, 1-11.https://doi.org/10.1007/s11270-016-2985-x.

24. Igamberdiev AU, Eprintsev AT. (2016). Organic acids: the pools of fixed carbon involved in

redox regulation and energy balance in higher plants. Frontiers in Plant Science 7: 1042.

25. Jewell, M. C., Campbell, B. C., & Godwin, I. D. (2010). Transgenic plants for abiotic stress

resistance. Transgenic crop plants, 67-132.https://doi.org/10.1007/978-3-642-04812-8_2.

26. Kabir, A. H. (2016). Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to

cope with chromium stress. Plant Biology, 18(4), 710-719. https://doi.org/10.1111/plb.12436

27. Kanto, U., Jutamanee, K., Osotsapar, Y., Chai-arree, W., &Jattupornpong, S. (2015). Promotive

effect of priming with 5-aminolevulinic acid on seed germination capacity, seedling growth and

antioxidant enzyme activity in rice subjected to accelerated ageing treatment. Plant Production

Science, 18(4), 443-454. https://doi.org/10.1626/pps.18.443.

28. Khatun, M. R., Mukta, R. H., Islam, M. A., & Huda, A. N. (2019). Insight into citric acid-induced

chromium detoxification in rice (Oryza sativa. L). International Journal of

Phytoremediation, 21(12), 1234-1240. https://doi.org/10.1080/15226514.2019.161962.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 872 www.rsisinternational.org

29. Kochian, L. V., Hoekenga, O. A., & Pineros, M. A. (2004). How do crop plants tolerate acid

soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annu. Rev. Plant

Biol., 55(1), 459-493.https://doi.org/10.1146/annurev.arplant.55.031903.141655.

30. Kong, X., Tian, H., Yu, Q., Zhang, F., Wang, R., Gao, S., ... & Ding, Z. (2018). PHB3 maintains

root stem cell niche identity through ROS-responsive AP2/ERF transcription factors in

Arabidopsis. Cell Reports, 22(5), 1350-1363.doi: https://10.1016/j.celrep.2017.12.105.

31. Koyama H., Ojima K., Yamaya T (1990). Utilization of anhydrous aluminum phosphate as a

sole source of phosphorus by selected carrot cell line. Plant physiol. 31:173-177.

32. Lichtenthaler, H. K., & Wellburn, A. R. (1983). Determinations of total carotenoids and

chlorophylls a and b of leaf extracts in different solvents. https://doi.org/10.1042/bst0110591.

33. Liujie Wu., Yuriko Kobayashi, Jun Wasaki, and Hiroyuki Koyama (2018). Organic acid

excretion from root: a plant mechanism for enhancing phosphorus acquisition, enhancing

aluminum tolerance, and recruiting beneficial rhizobacteria. Soil science and plant nutrition.

64(6): 697-704.

34. López-Bucio J, De la Vega OM, Guevara-García A, Herrera-Estrella L. (2000). Enhanced

phosphorus uptake in transgenic tobacco plants that overproduce citrate. Nature Biotechnology

18: 450–453.

35. Lutts, S., Kinet, J. M., &Bouharmont, J. (1996). NaCl-induced senescence in leaves of rice

(Oryza sativaL.) cultivars differing in salinity resistance. Annals of botany, 78(3), 389-

398.https://doi.org/10.1006/anbo.1996.0134.

36. Mahmud JA, Hasanuzzaman M, Nahar K, Borhannuddin Bhuyan MH, Fujita Masayuki(2018)

Insights into citric acid-induced cadmium tolerance and phytoremediation in Brassica juncea L.:

Coordinated functions of metal chelation, antioxidant defense and glyoxalase systems.

Ecotoxicology and Environmental Safety 147:990–1001.

https://doi.org/10.1016/j.ecoenv.2017.09.045

37. Martinoia, E.; Rentsch, D. Malate (1994) Compartmentation—Responses to a Complex

Metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol, 45: 447–467.

38. Meyer, S.; Scholz-Starke, J.; De Angeli, A.; Kovermann, P.; Burla, B.; Gambale, F.; Martinoia,

E. Malate (2011). Transport by the Vacuolar AtALMT6 Channel in Guard Cells Is Subject to

Multiple Regulation. Plant J. 67: 247–257.

39. Montiel-Rozas, M. D. M., Madejón, E., &Madejón, P. (2016). Effect of heavy metals and

organic matter on root exudates (low molecular weight organic acids) of herbaceous species: An

assessment in sand and soil conditions under different levels of contamination. Environmental

Pollution, 216, 273-281.https://doi.org/10.1016/j.envpol.2016.05.080.

40. Mourato M, Reis R, Martins LL(2012) “Characterization of plant antioxidative system in

response to abiotic stresses: a focus on heavy metal toxicity,” in Advances in Selected Plant

Physiology Aspects, G. Montanaro and B. Dichio, Eds., pp. 23–44, InTech, Vienna, Austria,

http://hdl.handle.net/10400.5/4410.

41. Mourato, M., Reis, R., & Martins, L. L. (2012). Characterization of plant antioxidative system

in response to abiotic stresses: a focus on heavy metal toxicity. Advances in selected plant

physiology aspects, 12, 1-17.

42. Moussa, Z., Judeh, Z. M., & Ahmed, S. A. (2019). Nonenzymatic exogenous and endogenous

antioxidants. Free radical medicine and biology, 1, 11-22.

https://doi.org/10.5772/intechopen.87778.

43. Mukta, R. H., Khatun, M. R., & Nazmul Huda, A. K. M. (2019). Calcium induces phytochelatin

accumulation to cope with chromium toxicity in rice (Oryza sativa L.). Journal of Plant

Interactions, 14(1), 295-302.https://doi.org/10.1080/17429145.2019.1629034.

44. Mukti, Gill. (2014). Heavy metal stress in plants: a review. Int J Adv Res, 2(6), 1043-1055.

45. Nath, K., Singh, D., Shyam, S., & Sharma, Y. K. (2008). Effect of chromium and tannery effluent

toxicity on metabolism and growth in cowpea (Vigna sinensis L. SaviexHassk)

seedling. Research in Environment and Life Sciences, 1(3), 91-94.

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 873 www.rsisinternational.org

46. Nematshahi, N., Lahouti, M., &Ganjeali, A. (2012). Accumulation of chromium and its effect

on growth of (Allium cepa cv. Hybrid). European Journal of Experimental Biology, 2(4), 969-

974.

47. Panda, S. K., & Choudhury, S. (2005). Chromium stress in plants. Brazilian journal of plant

physiology, 17, 95-102.https://doi.org/10.1590/S1677-04202005000100008.

48. Poonam Panchal, Anthony J. Miller, and Jitender Gir (2021).Organic acids: versatile stress-

response roles in plants.Journal of Experimental Botany. 72(11): 4038–4052.

49. Ramesh SA, Tyerman SD, Xu B, et al. (2015). GABA signalling modulates plant growth by

directly regulating the activity of plant-specific anion transporters. Nature Communications 6:

7879.

50. Rastgoo, L., Alemzadeh, A., &Afsharifar, A. (2011). Isolation of two novel isoforms encoding

zinc-and copper-transporting P1B-ATPase from Gouan (Aeluropus littoralis). Plant Omics

J, 4(7), 377-383.

51. Rauser, W. E. (1999). Structure and function of metal chelators produced by plants: the case for

organic acids, amino acids, phytin, and metallothioneins. Cell biochemistry and biophysics, 31,

19-48. https://doi.org/10.1007/BF02738153.

52. Reyes F, Pallas B, Pradal C, Vaggi F, Zanotelli D, Tagliavini M, Gianelle D, Costes E. (2020).

MuSCA: a multi-scale source-sink carbon allocation model to explore carbon allocation in

plants. An application to static apple tree structures. Annals of Botany 126: 571–585.

53. Riaz, A., Qin, Y., Zheng, Q., Chen, X., Jiang, W., Riaz, B., ... & Zeng, F. (2024). Cr (VI) behaves

differently than Cr (III) in the uptake, translocation and detoxification in rice roots. Science of

The Total Environment, 948, 174736.https://doi.org/10.1016/j.scitotenv.2024.174736.

54. Rolf D.V. inebrooke, Kathryn L, Cottingham, Jon Norberg, Marten Scheffer, Stanley I.

Dodson,Stephen C,Maberly, Ulrich Sommer (2004 Impacts of multiple stressors on biodiversity

and ecosystem functioning: The role of species co‐tolerance. Oikos, 104(3), 451-457.

https://doi:10.1111/j.0030-1299.2004.13255.x

55. Shanker, A. K., Cervantes, C., Loza-Tavera, H., &Avudainayagam, S. (2005). Chromium

toxicity in plants. Environment international, 31(5), 739-

753.https://doi.org/10.1016/j.envint.2005.02.003.

56. Shanker, A. K., Cervantes, C., Loza-Tavera, H., &Avudainayagam, S. (2005). Chromium

toxicity in plants. Environment international, 31(5), 739-

753.https://doi.org/10.1016/j.envint.2005.02.003.

57. Sharma, P., Jha, A. B., Dubey, R. S., &Pessarakli, M. (2012). Reactive oxygen species, oxidative

damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of

botany, 2012(1), 217037.https://doi.org/10.1155/2012/217037.

58. Sharma, S. S., & Dietz, K. J. (2006). The significance of amino acids and amino acid-derived

molecules in plant responses and adaptation to heavy metal stress. Journal of experimental

botany, 57(4), 711-726.https://doi.org/10.1093/jxb/erj073.

59. Solanki, R., & Dhankhar, R. (2011). Biochemical changes and adaptive strategies of plants under

heavy metal stress. Biologia, 66(2), 195-204.https://doi.org/10.2478/s11756-011-0005-6.

60. Szepesi, Á., & Szőllősi, R. (2018). Mechanism of proline biosynthesis and role of proline

metabolism enzymes under environmental stress in plants. In Plant metabolites and regulation

under environmental stress (pp. 337-353). Academic Press.https://doi.org/10.1016/B978-0-12-

812689-9.00017-0.

61. Thomas BK., David RP., Richard WZ. (2005). Organic acid secretion as a mechanism of

aluminum resistance: a model incorporating the cortex, epidermis, the external unstirred layer.

J. Exp. Bot. 56: 1853-1865.

62. Ullah, S., Liu, Q., Wang, S., Jan, A. U., Sharif, H. M. A., Ditta, A., ... & Cheng, H. (2023).

Sources, impacts, factors affecting Cr uptake in plants, and mechanisms behind

phytoremediation of Cr-contaminated soils. Science of the Total Environment, 165726.

https://doi.org/10.1016/j.scitotenv.2023.165726.

63. Viehweger, K. (2014). How plants cope with heavy metals. Botanical Studies, 55, 1-12.

https://doi.org/10.1186/1999-3110-55-35

INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51584/IJRSI | Volume X Issue X October 2025

Page 874 www.rsisinternational.org

64. Wang, A. S., Angle, J. S., Chaney, R. L., Delorme, T. A., & Reeves, R. D. (2006). Soil pH effects

on uptake of Cd and Zn by Thlaspi caerulescens. Plant and soil, 281, 325-

337.https://doi.org/10.1007/s11104-005-4642-9.

65. Weidinger, A., & Kozlov, A. V. (2015). Biological activities of reactive oxygen and nitrogen

species: oxidative stress versus signal transduction. Biomolecules, 5(2), 472-

484.https://doi.org/10.3390/biom5020472

66. Wong, H. L., Sakamoto, T., Kawasaki, T., Umemura, K., & Shimamoto, K. (2004). Down-

regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in

rice. Plant physiology, 135(3), 1447-1456.https://doi.org/10.1104/pp.103.036384.

67. Yokosho K, Yamaji N, Ma JF. 2011. An Al-inducible MATE gene is involved in external

detoxification of Al in rice. The Plant Journal 68:1061–1069.

68. Yu, G., Ma, J., Jiang, P., Li, J., Gao, J., Qiao, S., & Zhao, Z. (2019, August). The mechanism of

plant resistance to heavy metal. In IOP conference series: earth and environmental science (Vol.

310, No. 5, p. 052004). IOP Publishing.https://doi:10.1088/1755-1315/310/5/052004.

69. Zeng, F., Chen, S., Miao, Y., Wu, F., & Zhang, G. (2008). Changes of organic acid exudation

and rhizosphere pH in rice plants under chromium stress. Environmental Pollution, 155(2), 284-

289. https://doi.org/10.1016/j.envpol.2007.11.019

70. Zhang Y, Chen FS, Wu XQ, Luan FG, Zhang LP, Fang XM, Wan SZ, Hu XF, Ye JR. (2018).

Isolation and characterization of two phosphate-solubilizing fungi from rhizosphere soil of moso

bamboo and their functional capacities when exposed to different phosphorus sources and pH

environments. PLoS One 13: 1–14.

71. Zhou Y, Yang Z, Xu Y, Sun H, Sun Z, Lin B, Sun W, You J. (2018). Soybean NADP-malic

enzyme functions in malate and citrate metabolism and contributes to their efflux under Al stress.

Frontiers in Plant Science 8: 1–11

72. Ziad Moussa, Zaher MA, Judeh, Saleh A. Ahmed(2019) Nonenzymatic Exogenous and

Endogenous Antioxidants, In Rizwan Ahmad (Edited), Free Radical Medicine andBiology.

Intech Open.pp-1-22. DOI: 10.5772/intechopen.87778