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. 2024 Dec 8;30(12):2065–2075. doi: 10.1007/s12298-024-01536-4

Effect of aluminium toxicity on GI tagged Kachai lemon seedlings

Linthoingambi Ningombam 1, Budhindra Nath Hazarika 1,✉, Siddhartha Singh 2, Lobsang Wangchu 1, Nangsol Dolma Bhutia 3, Punabati Heisnam 4, Shubranil Das 1, Tabalique Yumkhaibam 3, KH Anush Sheikh 1
PMCID: PMC11685366  PMID: 39744319

Abstract

An experiment was performed to understand the effects of aluminium toxicity (AlCl3·6H2O) on Kachai lemon growth and development. The toxic effects of aluminium were assessed for 45 days in sand media. With untreated pots serving as the control, seedlings of 1 month old were exposed to three concentrations of AlCl3·6H2O: 300 μM, 600 μM and 900 μM. The nutrient Hoagland solution was also given to seedlings along with the Aluminium (Al) treatment. The outcome demonstrated that the chlorophyll content and carotenoids declined with the increase of the concentration levels of AlCl3·6H2O and interval of treatment. The contents of O2·− (Super oxide anion), H2O2 (Hydrogen peroxide) and OH (Hydroxyl radical) in seedlings increased with the higher concentration levels of aluminium and longer exposure to Al. Additionally, the activity of the enzymes catalase, superoxide dismutase, ascorbate peroxidase, peroxidase and glutathione reductase were increased in seedlings. Different non-enzymatic antioxidants’ actions like tocopherol and Vitamin C played important defence mechanisms for the maintenance of tolerance in aluminium toxicity by increasing their content with an increase in the concentration of treatment levels in Kachai Lemon.

Keywords: Acid soils, Aluminium toxicity, Enzymatic antioxidants, Kachai lemon, Non-enzymatic antioxidants, ROS, Total chlorophyll

Introduction

The current study was conducted in Kachai Lemon (Citrus jambheri Lush.). It is an exotic horticulture fruit native to the North East Indian state of Manipur. The Lemon is abundantly grown in the Kachai village of Ukhrul district, Manipur, and got a GI tagged (Geographical Indication) in 2014 and is locally known as Kachai Champra. Kachai Lemon has 45–51 mg ascorbic acid per 100 ml of juice and 36–56 ml of juice per fruit which is more than other lemon varieties. This research aims to comprehend the physiological and biochemical response of Kachai Lemon against Aluminium toxicity, AlCl3·6H2O was utilised in this work to imitate toxicity in plants. Aluminium toxicity in plants is frequently detected by morphological and physiological signs. The tolerance mechanism to Al toxicity is characterised by variances in root and shoot form and function. The negative effect of aluminium toxicity on numerous Citrus genotypes shows interference with reduced root respiration, calcium and phosphorus intake and utilization, and metabolic pathway, together with reduced root cell division. Citrus is sensitive to Aluminium toxic effects, thus it is impressive how these genotypes are able to withstand its harmful effects through different ways.

The toxicity of aluminium (Al) is a significant abiotic stress problem that many regions across the world confront. Aluminium is present in all soils; however it becomes harmful only in acidic soils with pH levels ranging from 4.5 to 5.5. Al is primarily found as insoluble deposits and is largely thought to be biologically inert in neutral or slightly acidic soils, but in acidic soils, Al3+ and other poisonous forms of Al are soluble in soil solutions and build up to large concentrations fast and started causing restriction of plant root growth through structural and functional damage to the roots, decreasing the uptake of nutrients and water, ultimately leading to subpar crop growth and output. Soil acidity affects 49 million hectares of land in India, with 25 million hectares having a pH less than 5.5. Such problematic soils can be found in the North Eastern states as well as some portions of the Western Ghats. Soils in India’s North Eastern states have a pH less than 5 and are considered very acidic in nature, accounting for Arunachal Pradesh (84%), Manipur (77%), Meghalaya (76%), Mizoram (50%), Sikkim (57%), and Tripura (47%) (Ishitani et al. 2004). Acidic soils are found mostly in the North Eastern region of India, as well as in a few other states such as UP, Bihar, JK, HP, MP, Karnataka, AP, Maharashtra and TN (Sharma and Roy 2022). The acidic character of soils occurs mostly in locations with high rainfall and varies based on soil texture, clay mineralogy, landscape geology, and buffering capacity. Acidification of the soil occurs naturally that is triggered and enhanced by certain agricultural practices. The likelihood of soil acidity is mostly due to nitrate leaching. Over 30% of the globe’s total area of land is covered by acidic soils (Chen et al. 2012). Acidic soil constraints imply a lower percentage of plant development and survival because roots are severely affected if pH value exceeds tolerance limits. Crops cultivated on acidic soils have a number of challenges, including the toxicity of aluminium, manganese, potassium, sulphur, nitrogen, boron, copper, and zinc.

Kachai lemon being grown in acidic soils and exhibits excellent quality as compared to other lemons therefore, investigation of the impact of Al stress is required in Kachai Lemon and no study has been conducted till date and the present work will be the maiden effort in understanding the stress responsive mechanism in Kachai lemon. Our study was primarily designed to investigate the short-term physiological and biochemical responses of Kachai lemon to Al exposure, with a focus on the initial stages of stress adaptation.

Materials and methods

Kachai lemon fruits were collected from Kachai village in Manipur’s Ukhrul District. The whole analysis of this research was conducted at Basic Science and Humanities lab of College of Horticulture and Forestry Pasighat. The fruit seeds were extracted, washed, removed from the pulp and treated fungicides. Then, on blotting paper in a bamboo frame, they were hydroponically germinated in an incubator at 25 ± 2 °C and 80% RH. Following germination, uniformly proportioned seedlings were transplanted into sterile river sand-filled pots made of clean imperforate plastic and given Hoagland nutritional solution, keeping its pH at 5.8. The seedlings were permitted to develop under laboratory conditions of College of Horticulture and Forestry in Pasighat, Arunachal Pradesh, under control conditions. Following that, Hoagland’s nutrient solution was applied to the pots having various concentrations of Aluminium chloride hexahydrate (AlCl3·6H2O), namely 0, 300 μM, 600 μM and 900 μM. In a completely randomised design, each of the treatment has 10 pots with 4 plants in each pot and replicated 3 times. Equal parts of treated Hoagland solution and sand were used for each of the pots. After every seven days, the solution was administered until the sand was saturated. The plants were uprooted after being exposed to the toxicity of aluminium-induced stress at three separate intervals of 15, 30, and 45 days (Fig. 1) and analysed. The 45 days duration was chosen to capture the critical early response phase of the plant to aluminum toxicity, allowing researchers to observe the enzymatic and non-enzymatic activity of Kachai lemon seedlings. Many plants exhibit an early burst of ROS production in response to abiotic stress, which serves as a signal to activate antioxidant defenses. Prolonged stress conditions can result in the depletion of antioxidant resources or cause damage to cellular structures, which can diminish the plant’s overall ability to respond effectively to the stress (Hartmann et al. 2022). By choosing a 45-day observation period, the study aimed to capture the plant’s response before reaching this critical point of resource depletion or irreversible damage, providing insights into the early defense mechanisms activated by the plant under aluminum stress.

Fig. 1.

Fig. 1

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Impact of the Aluminium (Al) toxicity on Kachai lemon seedlings at 15, 30 and 45 days after treatment application

Determination of leaf pigments

The method suggested by Lichtenthaler (1987) was used to estimate the total chlorophyll and carotenoid content of leaves. The amounts of pigments present were reported as mg g−1.

Determination of ROS

Measurement of superoxide anion (O2·−)

Determination of production of superoxide anion (O2·−) was done by measuring epinephrine autoxidation in terms of adrenochrome synthesis (Mishra and Fridovich 1972). Fresh root samples and shoot samples were cut into segments of 2–4 mm and deposited in reaction mixture of 2 ml comprising 20 mmol/L NaH2PO4 buffer of pH 7.8, 100 mol/L disodium-EDTA and 20 mol/L NADH. Initiation of reaction was done by adding 1.2 mmol/L of newly synthesised epinephrine (made in 0.1 M HCl). In a rotary shaker the samples were shaken at 150 rpm at 28 °C for 5–7 min and thereafter suitable aliquots were removed from the reaction mixture and measurement of absorbance change was carried out at 480 nm over the duration of 10 min. Formation of O2·− was measured in terms of ∆A [480 nm] min−1 g−1 fresh weight.

Measurement of hydrogen peroxide (H2O2)

Titanium sulphate was used to assess the production rate of H2O2 using spectrophotometric method (Jana and Choudhuri 1981). 150 mg of fresh shoots and roots samples were extracted using 3 ml of 50 mM sodium phosphate buffer (pH 6.5). The homogenate was centrifuged at 6000 × g for 15 min. Centrifugation 6000 × g for 15 min was repeated again after adding 1 ml of (0.1% titanium sulphate in 20% (w/v) H2SO4) in extracted solution. The supernatant’s yellow colour intensity was evaluated at 410 nm. H2O2 level was calculated using molar extinction coefficient 0.28 µM−1 cm−1 and was expressed as nmol g−1 tissue fresh weight.

Measurement of hydroxyl free radical (OH)

According to Halliwell and Gutteridge (1999), hydroxyl free radical (OH) generation was monitored. 100 g of fresh root or shoot samples were homogenised with 50 mM sodium phosphate buffer (pH 7.0) and centrifuged at 22,000 × g for 10 min at 4 °C. In test tubes, 1 ml of supernatant with 0.8 ml of 2.5 mM 2-deoxyribose and 0.2 ml of 2 mM FeSO4 was collected, properly mixed, and incubated in the dark for an hour. The reaction mixture was then treated with 1 ml of 0.25% thiobarbituric acid diluted in 10% trichloroacetic acid (TCA). The test tubes were then heated for 20 min before being instantly chilled in ice water for 10 min. The solution were then centrifuged at 3000 × g for 10 min, and the absorbance of the supernatant was measured in the spectrophotometer at 532 nm against a blank solution comprising 2 ml of 50 mM phosphate buffer and maintained pH at 7.0, 0.8 ml of 2.5 mM deoxyribose, and 0.2 ml of 2 mM FeSO4. The amount of hydroxyl free radical produced was calculated by multiplying the optical density of the supernatant by 103.

Determination of enzymatic antioxidants

Estimation of catalase (CAT) activity

The activity of catalase (CAT) was determined using the technique given by Beers and Sizer (1952). 50 mg of fresh shoot and root samples were dissolved in 2 ml of 50 mM Tris–HCl maintaining pH:8.0 adding 2% (w/v) PVP, 0.5 mM EDTA and 0.5% (v/v) Triton X-100 using a chilled pestle and mortar. Following dialysis, the resultant solution was centrifuged at 22,000 × g for 10 min at 4 °C, and the supernatant was used for enzyme testing. The test mixture contains 100 mM potassium phosphate buffer of pH: 7.0, 50 mM H2O2, and 200 µL enzyme extract in a total volume of 3 ml. The breakdown of H2O2 was followed by a 5 min decrease in absorbance 240 nm at 25 °C (extinction coefficient of 0.036 mM−1 cm−1). Catalase activity was expressed as µmol of H2O2 oxidized min−1 (mg protein)−1.

Estimation of superoxide dismutase activity (SOD)

SOD activity assessment was carried out by the method of Beauchamp and Fridovich (1971). Tissue samples (50 mg) from uprooted shoots/roots were macerated in 5 mL of K-phosphate buffer (50 mM; pH 7.8) using a chilled mortar and pestle. Centrifugation of the samples (10,000 rpm for 10 min) at 4 °C yielded the enzyme-rich supernatant. A 3 mL reaction mixture containing potassium phosphate buffer (50 mM; pH 7.8), EDTA (0.1 mM), Methionine (13 mM), Riboflavin (20 μM), NBT (100 μM) and 150 μL of crude sample was prepared. A spectrophotometer calibration was established through an enzyme-free NBT-free blank. A reference control containing only NBT (lacking enzyme) was also instituted. The tubes were exposed to 400 W light (4 × 100 W bulbs) for 15 min and absorbance was measured at 560 nm using an UV–Visible spectrophotometer (Model: AVI-2704). One unit of SOD activity is defined as the amount of enzyme required to cause 50% inhibition of the rate of NBT reduction measured at 560 nm. The amount of SOD is expressed as unit mg−1 protein.

Estimation of ascorbate peroxidase activity (APX)

The method developed by Nakano and Asada (1981) was utilized to determine APX activity. 100 mg root and shoot samples were crushed in 2 ml of 50 mM K phosphate buffer of pH 7.8 including 1% (w/v) PVP, 1 mM AsA, and 1 mM PMSF and centrifuged for 15 min at 4 °C at 15,000 × g. The reaction mixture contains 50 mM K-phosphate buffer (pH level 7.0), 0.2 mM EDTA, 0.5 mM AsA, and 1 mM H2O2 in a total volume of 3 ml. After H2O2-dependent reduction of AsA, a decrease in absorbance at 290 nm (extinction coefficient of 2.8 mM−1 cm−1) was recorded at 30 s intervals for up to 5 min. Enzyme specific activity is expressed as μmol ascorbate oxidized min−1 mg−1 protein.

Estimation of peroxidase (POD)

The approach described in Wang et al. (2015) was used to measure POD activity. 0.5 g seedling samples were homogenized using a cooled mortar and pestle containing 5 mL of 50 mM phosphate buffer of pH 5.5 and 0.2 g PVP. The mixture was centrifuged at 3000 × g at 4 °C for 10 min. 0.1 mL of supernatant was blended with 1 mL of 50 mM guaiacol, 2.9 mL of 50 mM phosphate buffer (pH 5.5), and 1 mL of 2% (v/v) H2O2 in a water bath at 37 °C. After 5 min, the absorbance at 470 nm was measured using a spectrophotometer. A unit of enzyme activity (U) was defined as a change in absorbance of 0.01 units per hour.

Estimation of glutathione reductase (GR) activity

GR activity was analysed using a modified version of Schaedle and Bassham (1977). 100 mg shoot and root samples were crushed in 2 ml of 100 mM Tris–HCl buffer of pH 7.8 at 4 °C using a chilled mortar and pestle. After centrifugation for 20 min at 4 °C at 22,000 × g, 3 ml of the supernatant was collected as reaction mixture and added 100 mM Tris–HCl buffer of pH 7.8, 0.5 mM oxidised glutathione (GSSG), 0.2 mM NADPH, 0.5 mM MgCl2, and 200 µl of enzyme extract utilised for enzyme testing. The assays were started by adding NADPH at room temperature. The reaction was measured by analyzing the decrease in NADPH absorbance at 340 nm (extinction coefficient 6.2 mM−1 cm−1) for 5 min. The enzyme specific activity was expressed in µmol NADPH oxidized min−1 mg−1 protein.

Determination of non-enzymatic antioxidants

Estimation of AsA (Ascorbic acid)

The ascorbic acid concentration was determined using the spectrophotometric method reported by Jagota and Dani (1982). In a clean mortar and pestle, 50 mg of root or shoot samples were macerated in 1.2 mL of 6% metaphosphoric acid. Following adequate maceration, the volume was increased to 2 ml using 3% metaphosphoric acid. For 10 min, the solution was centrifuged at 5000 rpm. 200 µl of the supernatant was taken out in test tubes. For the standard curve, different aliquots of ascorbic acid standard working solution were produced in another set of tubes. The volumes of all test tubes were then filled to 1 ml with 3% metaphosphoric acid, and 3 ml distilled water was added to each. Folin’s reagent was added to the aliquot and thoroughly mixed. A 10-min incubation period at room temperature was provided. The solution then turned blue and the absorbance at 760 nm was measured.

Data analysis

Completely randomized design (CRD) was used for all studies, including treatment, growth, and evaluation of biochemical parameters. Each treatment consisted of ten pots and was replicated three times. Analysis of variance (ANOVA) was used to assess the results, and means were separated by the least significant difference at P < 0.05 (Gomez and Gomez 2010). The results were presented in the form of mean ± SE (standard error). Thereafter, the comparison among the treatment levels was done by assessing the percentage decrease or increase (depending upon the parameter) in the values of the treated samples with respect to the values of their controls. Duncan’s Multiple Range Test (DMRT) was applied using WASP 2 software (ccari.icar.gov.in) to compare the means of different treatments. This post-hoc test allows for pairwise comparisons while controlling the Type I error rate. A significance level of 0.05 was used, and groups that do not differ significantly are considered statistically at par. The results are presented with letter groupings (e.g., a, b, c, d) to indicate the significance levels of the differences between the means. If two means are both labeled “a” or same letter they are considered statistically similar, or “statistically at par”.

Result and discussion

Leaf pigments

One of the most important analysis which must be done when plants are under environmental stress is to check the chlorophyll pigments in leaves as it will be the indicator of plants sickness (Parekh et al. 1990). In this experiment, Kachai lemon seedlings started showing sufficient decrease in the leaf pigments with higher declined under higher concentrations overtime as shown in (Fig. 2). Chlorophyll (Chl) a and b, total chlorophyll and carotenoid decreases with increase in concentration levels of Al and highest at severe stress, Al exposure of 900 µm at 45 days. In 15 days analysis of Chl a and total chlorophyll, it was observed that Al treatment of 300 µm is statistically at par with Al 600 µm. In Chl b, all the Al treatments at 45 days are found significant, 30 days all Al treatments are statistically at par and at 15 days, Al 300 µm is statistically at par with Al 600 µm and Al 900 µm is statistically at par with Al 600 µm. The decrease in pigments contents is due to the degradation of chlorophyll pigments under Al stress conditions. In support to this study (Santos 2004) and (Smirnoff 1996) suggested that under stress conditions an enzyme called chlorophyllase is activated and inhibits the functions of chlorophyll which ultimately leads to degradation.

Fig. 2.

Fig. 2

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Changes in Chlorophyll a, Chlorophyll b, Total chlorophyll and Total carotenoids of Kachai lemon subjected to Aluminium (Al) treatments. Values represent the mean ± SE (n = 3)

ROS production

Reactive oxygen species like superoxide anion (O2·−), hydroxyl radical (OH) as well as like hydrogen peroxide (H2O2) and singlet oxygen (1O2) are the unwanted free radicals generated during aerobic metabolisms of plants and are found in chloroplasts, peroxisomes and mitochondria. These species become harmful if present at higher concentrations (Takagi et al. 2016). These are produced more when plants are under environmental stress conditions. The Al exposure resulted in higher levels of OH radical, O2·− and H2O2 in both shoots and roots (Fig. 3). However, during the Al treatment in roots, the generation of ROS was found highest at 45 days at severe stress level (Al 900 µM). In roots at 45 days generation of H2O2 was found non-significant among the three Al treatment levels. But in shoots, generation of O2·− and H2O2 were consistently higher in 15 days than 45 days except OH radical showing the adaptive response of Kachai lemon seedlings. The H2O2 generation in shoots subjected to exposure of all Al treatments at 15 days was found non-significant. The increase in ROS under aluminum toxicity in Citrus species is due to a combination of disrupted cellular processes, impaired photosynthesis and respiration, inhibition of antioxidant defenses, and resultant oxidative stress. The elevated generation of ROS leads to cell death by attacking the cell membrane, lipids peroxidation, and inactivation of enzyme for protein (Srivastava and Dubey 2011). Similar study was done by Xu et al. (2012) and reported that Al-sensitive genotype increased more ROS levels in wheat than tolerant genotype.

Fig. 3.

Fig. 3

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Generation of ROS (Reactive oxygen species) H2O2 (Hydrogen peroxide), OH (Hydroxyl radical) and O2·− (Superoxide anion) in shoots (a) and roots (b) subjected to Aluminium (Al) treatments in Kachai lemon. Values represent the mean ± SE (n = 3)

Enzymatic antioxidant tolerance mechanisms

The highly reactive and damaging ROS generated profusely under stress conditions attack nucleic acids, lipids, and proteins, ultimately resulting in cell death if not balanced (Sachdev et al. 2023). Hydrogen peroxide (H₂O₂) and superoxide (O₂−) are among the most common ROS, playing dual roles in plant cells both as signal defense responses and indicators of oxidative stress (Smirnoff and Arnaud 2019). SOD is crucial in the first line of defense against oxidative stress, rapidly converting the superoxide radical (O₂−) into hydrogen peroxide (H₂O₂) and molecular oxygen (O₂) thereby preventing oxidative damage (Zheng et al. 2023). CAT is responsible for breaking down hydrogen peroxide into water and oxygen, a critical step in reducing oxidative stress. By upregulating CAT activity, plants can effectively detoxify ROS, thereby preventing cellular damage and maintaining cellular homeostasis (Ibrahim et al. 2018). GR plays a pivotal role in maintaining the pool of reduced glutathione (GSH) by converting oxidized glutathione (GSSG) back into its reduced form. This regeneration of GSH is essential because GSH acts as a powerful antioxidant, either directly scavenging ROS or serving as a cofactor for other antioxidant enzymes (Csiszar et al. 2016). Peroxidase helps protect cell membranes by reducing lipid peroxidation, thereby protecting membrane integrity (Farooq et al. 2009). In Kachai lemon, the activities of CAT, SOD, APX, POD and GR were consistently showing higher with highest et al. 900 µM in shoots and roots showing the plants adaptive response similar to many previous studies and all the Al treatments were found significantly different (Fig. 4). But in CAT enzyme activity at 30 days, control and Al 300 µM were statistically at par in shoots. CAT activity did not change at 300 µM Al concentration suggests that the level of H₂O₂ might not have reached a threshold that necessitates an increased CAT response. Alternatively, the unchanged CAT activity could indicate that other enzymes, particularly APX, might be compensating for H₂O₂ detoxification, thereby reducing the demand for catalase activity. This finding aligns with previous research, such as the study by Boscolo et al. (2003) which reported a similar lack of increase in CAT activity under Al-induced oxidative stress in maize. In SOD activity, Al 600 and 900 µM were statistically at par in both 15 and 30 days interval in roots however at 45 days, Al 300 and 600 µM were statistically at par. In POD enzyme activity Al treatments of 600 and 900 µM were statistically at par in 45 days root analysis and Al 300 and 600 µM were statistically at par in 30 days shoot analysis. In GR activity analysis, it was observed that in shoots at 15 and 45 days analysis, Al treatments of 300 and 600 µM were at par. In 15 days, increasing percentage of CAT and POD enzyme activity compared to control in both shoots and roots were at peak which then reduced at 30 and 45 days. However, in SOD and APX, the enzyme activity was found highest in Al 900 µm at 45 days which gradually shows increasing starting from 15 days. The increase in SOD activity suggests that there was a significant production of superoxide radicals (O₂−) in response to aluminum stress. SOD is the first line of defense against oxidative stress, converting O₂− into H₂O₂ (Zheng et al. 2023). The elevated SOD activity reflects an adaptive response to prevent the accumulation of harmful superoxide radicals. The increased APX activity indicates that the plant cells are actively detoxifying the H₂O₂ produced by SOD (Li 2023). The significant increase in APX activity under 300 µM Al stress could also indicate that APX is taking over the role of H₂O₂ detoxification, thereby reducing the need for an increase in CAT activity (Boscolo et al. 2003). In GR enzyme activity compared to control, in shoots the enzyme activity gradually decreases with longer duration of exposure but in roots enzymatic activity increases with higher stress levels overtime (Fig. 4). The increase in enzymatic activity reflects the plant’s effort to maintain a balance between ROS production and scavenging (Zhang et al. 2020). The difference in the activity of enzymes tolerating the oxidative stress is because different plant species and tissues have different response mechanisms (Boscolo et al. 2003). The differential response in these enzyme activities could also be regulated by specific signaling pathways activated under aluminum stress like transcription factors or signaling molecules may selectively upregulate SOD and APX without a corresponding increase in CAT, depending on the specific ROS generated and the localization of these ROS within cellular compartments. Similar research of toxicity induced by Al treatments in many citrus species had been already done by Chen et al. (2005) in Cleopatra mandarin (Citrus reshni); (Li et al. 2016) in Citrus grandis and Citrus sinensis seedlings and (Riaz et al. 2018) in Trifoliate orange (Poncirus trifoliate).

Fig. 4.

Fig. 4

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Changes of the OH (Hydroxyl radical) in shoots (a) and roots (b) subjected to Aluminium (Al) treatments in Kachai lemon. Values represent the mean ± SE (n = 3)

Non-enzymatic antioxidant tolerance mechanisms

Aluminum toxicity leads to the overproduction of reactive oxygen species (ROS), which can initiate lipid peroxidation, damaging cell membranes. Tocopherol (Vitamin E), being a lipophilic antioxidant, is particularly effective in protecting cell membranes. It intercepts lipid radicals, preventing the propagation of lipid peroxidation. This action helps maintain the integrity of cellular membranes, which is crucial under stress conditions like aluminum toxicity (Ghosh et al. 2022). Tocopherol works in synergy with other antioxidants, particularly AsA (van Doorn and Ketsa 2014). When tocopherol neutralizes a lipid radical, it becomes oxidized to a tocopheroxyl radical. AsA can regenerate tocopherol from this radical form, restoring its antioxidant capability. This regeneration cycle is vital for sustaining the antioxidative defense over extended periods of stress (Szarka et al. 2012). Ascorbic acid acts as a primary ROS scavenger in both the cytosol and chloroplasts. It serves as a substrate for ascorbate peroxidase (APX) in the ascorbate–glutathione cycle, helping to detoxify hydrogen peroxide (H2O2). The synergistic action of tocopherol and ascorbate is central to maintaining redox homeostasis in Kachai lemon under aluminum stress. This division of labor ensures that ROS are effectively neutralized in different cellular compartments. Aluminium treatments started altering ascorbic acid content in Kachai lemon shoots and roots from 15 days and found gradually increasing the non- enzymatic antioxidant content at 30 and 45 days (Fig. 5). AsA content was highest et al. 900 µM at 45 days in both shoots and roots. AsA content in roots at 15 days Al 300 µM was statistically at par with Al 600 µM and in shoots at 15 and 30 days, control and Al 300 µM were at par (Fig. 5). The increase in non-enzymatic antioxidants like AsA in Citrus species under higher stress levels over time is a critical adaptive response to oxidative stress. This antioxidant play a key role in scavenging ROS, protecting cellular structures, and maintaining redox homeostasis, which helps the plant survive and thrive under adverse conditions (Zhou et al. 2015).

Fig. 5.

Fig. 5

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Influence of aluminium on Ascorbic acid content in Kachai lemon shoots (a) and roots (b) (µg/g fresh weight)

Conclusion

Toxicity of aluminium is very hazardous to plants growing in acidic environments, even in little doses. Citrus fruit crops are particularly vulnerable to Al toxicity, and the Kachai lemon is a GI-tagged fruit crop that grows in the acidic hills of Manipur. Aluminium toxicity poses significant challenges to Kachai lemon cultivation, affecting its photosynthetic machinery and inducing oxidative stress. However, the plant’s elaborate defensive mechanisms, both enzymatic and non-enzymatic, play a pivotal role in alleviating the detrimental effects of Aluminium toxicity. This is the first study attempt to understand the physiological and biochemical reactions of Kachai lemon under Al stress conditions, as well as the identification of stress tolerance pathways in Kachai lemon and the present findings provide a foundation for further studies aiming to enhance the tolerance of citrus plants under aluminium stress conditions, potentially through biotechnological advancements or breeding programs.

Acknowledgements

I would like to thank my advisory committee and my classmate who are authors here for their guidance and contribution in this research work. I would like to extend my sincere thanks to College of Horticulture and Forestry, CAU (I), Pasighat, Arunachal Pradesh campus for providing essential facilities during the course of this research.

Author contributions

In this study, all writers produced equivalent amounts of effort. My advisors Siddhartha Singh, Lobsang Wangchu, and Nangsol Dolma Bhutia, as well as my supervisor B.N. Hazarika, helped in outlining the research work and discussing the treatment information to be used in this study. Linthoingambi Ningombam prepared the planting material for investigation and data collecting and statistical analysis. The sand medium for growing the Kachai lemon seedlings was prepared with assistance from KH. Anush Sheikh and Tabalique Yumkhaibam. Shubranil Das contributed in arranging references of the manuscript. Under the guidance of Punabati Heisnam, Linthoingambi Ningombam wrote the manuscript’s initial draft, and all of the contributors offered feedback on earlier drafts. The final manuscript was reviewed and approved by all writers.

Declarations

Conflict of interest

There are no financial or non-financial interests of the authors to report.

Footnotes

Publisher's Note

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