Introduction

Wheat (Triticum aestivum L.) is one of the the oldest and most widely cultivated cereal crop, playing a key role in global food security1. It is grown in irrigated, tropical and subtropical regions due its adaptability to a wide range of climatic conditions2. Pakistan ranked 4th amongst Asian countries and 11th in the world regarding wheat production with an annual production about 24 million tons1. Wheat is primarily consumed as “Chapati” (bread) locally, and is used for making bread, biscuits, sweets, noodles, gluten, alcoholic beverage, cosmetics and its protein is used as meat substitute3. Salinity is a major constraint to agricultural production, limiting crop yields. It has affected the about 33% of irrigated and 20% of cultivated area worldwide4 and about 30% of the total irrigated area in Pakistan5. Saline areas are increasing at a rate of 10% annually6 whereas total salt affected area is supposed to increase around 25% by 20507,8 potentially could have severe consequences for geographical distribution of plant communities.

Salt stress in plants impacts the synthesis, transport and utilization of carbohydrates for tissues formation9. It disrupts physiological processes such as osmotic adjustment, ionic toxicity and nutritional variation, while decreasing external water potential, water uptake and cell expansion10. It also leads to stomata closure and reduced the ability of the plants to assimilate CO211,12. Furthermore, Salt stress led to the over production of reactive oxygen species (ROS) in plant tissues13 which results oxidative damage to cellular macromolecules and disrupt essential cellular function in plants 14.

Gamma amino butyric acid (GABA) is an important four carbon, non-protein amino acid which regulates the plant growth by involving certain physiological and biochemical processes15. It maintains cytosolic pH, carbon/nitrogen flux for tricarboxylic acid cycle, osmotic pressure and regulation of redox status16. Its exogenous application enhanced the photosynthetic activities, enzymatic and non-enzymatic reactions, and membrane stability under stress conditions17. Previously GABA-induced regulations in growth and physio-biochemical processes in plants under stress conditions were well reported18.

Exogenous GABA application has been found to improve the salt stress tolerance in wheat plants due to the enhancement of nitrogen and carbon assimilation, photosynthesis and antioxidant enzyme activities by elevating resistance to oxidative stress19,20,21. GABA could also promote the production of proline, soluble sugar, and other osmotic regulating substances to protect the wheat seedling from oxidative damage under drought stress22. However, GABA induced modification in growth and anatomical features of wheat under salt stress were rarely investigated. Therefore, the present study was aimed to examine exogenous GABA induced salinity tolerance by modulating early growth and morpho-anatomical features in wheat.

Results

Morphological characters

Plant height (cm)

The GABA treatment enhanced the plant height by 2.49, 6.58 and 2.84% in FSD-08, and 1.79, 3.22 and 3.22% in ANAJ-17 at 1 mM, 2 mM and 3 mM, respectively under saline conditions (Fig. 1A).

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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Effect of exogenous GABA application on (A) plant height, (B) spike length, (C) shoot length, and (D) root length under saline and non-saline conditions. Vertical bars with different lowercase letters above are significantly different at P ≤ 0.05 by LSD Tests. Capped bars represent SD (n = 4). 0, 1, 2, 3 mM are GABA concentrations.

Spike length (cm)

In FSD-08, spike length was improved by 1.78, 6.04, and 3.80%, whereas it was significantly increased in ANAJ-17 by 9.36, 4.88 and 4.48% at 1 mM, 2 mM and 3 mM respectively (Fig. 1B).

Shoot length (cm)

Exogenous GABA application enhanced shoot length in FSD-08 by 5.07, 8.45 and 7.18% while 2.79, 1.86 and 2.56% at 1 mM, 2 mM and 3 mM, respectively in ANAJ-17 under saline condition (Fig. 1C).

Root length (cm)

Root length with GABA treatment was increased upto 9.18% for ANAJ-17 and 4.75% for FSD-08 under saline condition (Fig. 1D).

Awn length (cm)

Moreover, under non-saline conditions, the awn length was enhanced by 14.70, 12.35 and 10.58% in ANAJ-17 as compared to FSD-08 at 1 mM, 2 mM and 3 mM GABA application, respectively (Fig. 3A).

Shoot fresh and dry weight (g)

In addition, GABA showed substantial increase in shoot fresh weight by 8.73% at 1 mM, 18.51% at 2 mM and 4.08% at 3 mM in FSD-08, whereas, in ANAJ-17 by 17.36% at 1 mM, 13.26% at 2 mM and 10.08% at 3 mM under saline condition (Fig. 2A).

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
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Effect of GABA on (A) shoot fresh weight, (B) shoot dry weight, (C) root fresh weight, and (D) root dry weight under saline and non-saline conditions. Vertical bars with different lowercase letters above are significantly different at P ≤ 0.05 by LSD Tests. Capped bars represent SD (n = 4). 0, 1, 2, 3 mM are GABA concentrations.

Substantial improvements in shoot dry weight i.e., 21.34, 22.58 and 5.95% in FSD-08 and 15.14, 1.26 and 5.83% in ANAJ-17 were noted at 1 mM, 2 mM and 3 mM GABA levels, respectively under saline condition (Fig. 2B).

Root fresh and dry weight (g)

Root fresh weight was enhanced by 54.65, 32.49, and 4.75 50.12% at 1 mM, 2 mM and 3 mM, respectively in FSD-08, and 3.23% at 1 mM in ANAJ-17 (Fig. 2C).while root dry weight was enhanced by 39.51% at 1 mM and 3.66% at 3 mM in ANAJ-17 under non-saline condition (Fig. 2D).

Leaf area (cm2)

The leaf area was enhanced by 26.57, 4.38 and 10.57% in ANAJ-17 as compared to FSD-08 under saline conditions (Fig. 3B).

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Effect of GABA on (A) awn length, (B) leaf area, (C) no of roots and (D) no of leaves under saline and non-saline conditions. Vertical bars with different lowercase letters above are significantly different at P ≤ 0.05 by LSD Tests. Capped bars represent SD (n = 4). 0, 1, 2, 3 mM are GABA concentrations.

Number of leaves

The number of leaves were comparatively higher in GABA treated plants than non-treated plants under salt stress conditions (Fig. 3D).

Number of roots

The number of roots was 2.70, 8.10 and 5.40% higher at 1 mM, 2 mM and 3 mM, respectively in ANAJ-17 as compared to FSD-08 under saline conditions (Fig. 3C).

Stem anatomical characters

GABA-induced stem anatomical modifications are following:

Stem radius (µm)

GABA application substantially increased the stem radius by 1.19% at 1 mM, 4.19% at 2 mM and 7.53% at 3 mM in ANAJ-17 as compared to FSD-08 under saline condition (Table 1).

Table 1 GABA induced modification of stem anatomical parameters of wheat cultivars under saline and non-saline conditions.
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Epidermal thickness (µm) and epidermal cell area (µm2)

GABA application enhanced the epidermal thickness by 43.30, 33.33 and 60.88%, whereas, epidermal cell area by 14.81, 7.40 and 18.51% at 1 mM, 2 mM and 3 mM, respectively in FSD-08 as compared to ANAJ-17 under saline conditions (Table 1).

Sclerenchyma thickness (µm) and sclerenchyma cell area (µm2)

Moreover, the GABA application showed significant increase in sclerenchyma thickness by 10.66, 21.55 and 26.66% at 1 mM, 2 mM and 3 mM, respectively in FSD-08 whereas in ANAJ-17 upto 63.26% under saline condition. Under non-saline condition, the GABA showed significant increase in sclerenchyma cell area by 18.91, 37.43 and 25.67% in FSD-08 while in ANAJ-17 by 71.31, 66.66 and 56.28% at 1 mM, 2 mM and 3 mM, respectively (Table 1).

Phloem cell area (µm2)

Furthermore, GABA application showed significant improvement in phloem cell area by 62.06, 24.03 and 49.18% in FSD-08 while in ANAJ-17 by 16.38, 41.26 and 61.34% at 1 mM, 2 mM and 3 mM respectively under saline conditions (Table 1).

Metaxylem cell area (µm2)

In FSD-08, metaxylem cell area was improved significantly by 29.47, 11.83 and 13.35% while in ANAJ-17 by 10.80, 8.57 and 14.28% at 1 mM, 2 mM and 3 mM respectively under non-saline condition (Table 1).

Vascular bundle area (µm2)

Moreover, vascular bundle area was enhanced significantly in FSD-08 by 36.90 at 1 mM, 6.86 at 2 mM and 17.59% at 3 mM under non-saline condition (Table 1).

Root anatomical characters

GABA-induced root anatomical modifications are these:

Epidermal thickness (µm) and epidermal cell area (µm2)

GABA application significantly increased the epidermal thickness by 4.54, 17.06 and 31.81%, and epidermal cell area by 36.42, 18.21 and 43.34% at 1 mM, 2 mM and 3 mM respectively in ANAJ-17 as compared to FSD-08 under saline condition (Table 2).

Table 2 GABA induced modification of root anatomical parameters of wheat cultivars under saline and non-saline conditions.
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Cortical thickness (µm) and cortical cell area (µm2)

The cortical thickness was increased by GABA application i.e. 23.45 at 1 mM, 20.98 at 2 mM and 25.92% at 3 mM in FSD-08 and by 9.30% in ANAJ-17 at 2 mM under saline condition. However, cortical cell area was enhanced significantly by 8.15, 37.39 and 12.80% at 1 mM, 2 mM and 3 mM respectively in FSD-08 while in ANAJ-17 by 7.30% at 2 mM under saline condition (Table 2).

Endodermal thickness (µm) and endodermal cell area (µm2)

The endodermal thickness was not improved in FSD-08, whereas, in ANAJ-17 it was increased by 7.69% at 2 mM under saline condition. In ANAJ-17, the GABA application improved the endodermal cell area significantly by 4.95 at 1 mM, 3.03 at 2 mM and 27.27% at 3 mM as compared to FSD-08 under saline condition (Table 2).

Pith radius (µm) and pith cell area (µm2)

Furthermore, GABA significantly improved the pith radius by 41.66 at 1 mM, 26.66 at 2 mM and 21.66% at 3 mM in FSD-08, and in ANAJ-17 by 9.80 at 1 mM and 11.76% at 2 mM under saline condition. While pith cell area was enhanced by 57.70, 48.23 and 31.47% in FSD-08 at 1 mM, 2 mM and 3 mM, respectively (Table 2).

Metaxylem cell area (µm2)

Under non-saline condition, GABA application significantly improved the metaxylem cell area by 16.87, 26.23 and 54.88% (Table 2).

Phloem cell area (µm2)

Phloem cell area was increased by 17.68, 24.21 and 16.08% in ANAJ-17 under non-saline condition (Table 2).

Discussion

GABA is an integral component of natural plant metabolism, playing a key role in signaling mechanisms. It serves as an important intermediate in nitrogen metabolism and amino acid biosynthesis. Its dual function as both a metabolite and signaling pathways enables plants to cope with abiotic stresses23. Exogenous GABA application was found to significantly improve the morphological traits of wheat plants of both cultivars, FSD-08 and ANAJ-17. This improvement included increased plant height, shoot fresh and dry weight, root and shoot length, spike length, number of root and leaves, and leaf area under salt stress (Figs. 1, 2, and 3). Previous research has shown that the salt stress substantially impacts the activity of antioxidant enzymes, photosynthetic pigments, and the ionic balance in maize24. GABA application has been reported to regulate the antioxidant enzyme activities to improve yield and quality of fragrant rice25,26. The level of endogenous GABA was increased in both rice27 and tomato28 under salt stress. In strawberry plant, salinity tolerance was improved through the activation of salinity related genes transcription and enhancing enzymatic and non-enzymatic antioxidant physiological response upon GABA application29.

Exogenous GABA application significantly increased the shoot fresh and dry weight of both wheat cultivars, FSD-08 and ANAJ-17, under saline condition (Fig. 2A, B). Previous studies have shown that GABA boost shoot biomass by promoting the cell division and expansion, thus the maintaining metabolic balance in carrots30. Additionally, GABA application enhances the biomass and improves the nutrition value in germinating soybeans under salt stress31. It has also been reported to significantly increase the shoot fresh weight of maize seedlings17, Vigna mungo at 70 mM32, enhance shoot dry weight in wheat under salt stress 33. GABA application substantially improved the plant height, spike length and shoot length of FSD-08 and ANAJ-17 under saline condition (Fig. 1A, B, C). Previous research suggests that GABA induced improvements in plant growth are linked to increase net photosynthetic rate, gas exchange capacities as well as the improved antioxidant enzyme activities to scavenge ROS34. Moreover, the GABA application has been shown to significantly enhance the morphological and physio-biochemical responses of fragrant rice35.

Additionally, GABA application significantly improved the root length of both wheat cultivars, FSD-08 and ANAJ-17 under saline condition (Fig. 1D). Its application increased the root length to enhance the uptake of water and nutrients36, and for the absorption of more nitrate in lettuce37. GABA also improved the root length in maize17 and carrot38 under slat stress by altering ionic balance. The application of GABA substantially enhanced the leaf area and number of leaves of both cultivars, FSD-08 and ANAJ-17 under saline condition (Fig. 3B, D). In rice, GABA application improved the nutrient uptake and proline content in leaves39. The enhanced growth and stress tolerance observed in maize were attributed to increase chlorophyll content and an effective antioxidant defense system40 which protect the chloroplasts and PSII function from the harmful effects of salt41. Furthermore, GABA application significantly increased the number of leaves and leaf area in Vigna mungo under salt stress42 to allowing for greater sunlight capture and improved photosynthetic rate43.

Under non-saline conditions, GABA application also significantly improve the plant growth also by enhancing nutrient uptake particularly potassium and nitrogen assimilation44. Its application substantially improved root fresh and dry weight in ANAJ-17 under non-saline condition (Fig. 2C, D). Previous studies had shown that GABA up regulated net photosynthesis rate, anti-oxidant enzymes and nitrogen metabolism to enhance the root fresh and dry weight of maize seedlings24. Furthermore, GABA application improved the root fresh and dry weight in soyabean45, maize seedlings46 and cotton by stimulating cell division through the activation of GABA receptors and downstream signaling pathways47,48.

Under non-saline conditions, GABA also significantly enhanced plant growth of two wheat cultivars, FSD-08 and ANAJ-17, by anatomical features such as stem radius, epidermal thickness, cell area, sclerenchyma thickness, and phloem area (Table 1). While root and pith radius, epidermal thickness and its cell area, endodermal cell area, sclerenchyma and cortical thickness was increased significantly under saline condition (Table 2). These anatomical changes may be due to reduced cell elongation, restricted cell division and alterations in cell differentiation49.

Research on GABA-induced changes in the wheat anatomy has been relatively limited, and no specific information was found in this area. However, in present study, the GABA application significantly increased the root endodermal and epidermal thickness of ANAJ-17 under saline condition (Fig. 5). The increased endodermal thickness prevents water loss and alters the ion transport in the maize under salt stress50. It also restricts entry of excessive Na+ and Cl− into cells and serves as a barrier to prevent the leakage of K+ ions and water51. Previous studies revealed that plant growth regulators significantly stimulated root epidermal thickness of wheat52, epidermal and endodermal thickness of radish seedling under salt stress53.

Furthermore, GABA application notably increased the stem metaxylem and phloem area of both wheat cultivars under non-saline and saline condition (Fig. 4). Studies have indicated that GABA enhances the phloem area, improving the nutrients transport into healthy cells for various metabolic processes54. The significant increase in metaxylem area is an adaptive mechanism to ensure consistent water flow and mineral ions transport under saline condition in Panicum55. Previous research has shown that plant growth regulator stimulated metaxylem and phloem area of radish seedling under salt stress56. Additionally, GABA application substantially reduced the root cortical thickness of both wheat cultivars under non-saline condition (Fig. 5). This reduction in cortical thickness is likely due to the collapse of cortical cells, which helps conserve essential energy for survival and growth of plants under salinity stress57. Studies have shown that plant growth regulators reduce root cortical thickness of Purslane under salt stress58.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
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Transverse section of stem at different concentration of GABA under saline and non-saline conditions. GABA application increased stem metaxylem and phloem area improve nutrient transport and metabolic pathways of both wheat cultivars under non-saline and saline conditions. EP: Epidermis, Sc: Sclerenchyma, Xy: Xylem, Ph: Pholem, VB: Vascular Bundle.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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Transverse section of root at different concentration of GABA under saline and non-saline conditions. GABA application substantially increased the root endodermal and epidermal thickness of ANAJ-17 under saline condition which prevents entry of excessive salts. EP: Epidermis, Sc: Sclerenchyma, Cor: Cortex, End: Endodermis, Pi: Pith, Xy: Xylem, Ph: Pholem, VB: Vascular Bundle.

Conclusion

Overall, the application of exogenous GABA enhanced the early growth, including improvements in plant height, shoot fresh and dry weight, root length and leaf area and anatomical features such as root epidermal thickness and cell area, pith radius and endodermal thickness under saline conditions. Among the cultivars, the ANAJ-17 performed better than FSD-08. In conclusion, foliar GABA application improved the morpho-anatomical features of wheat. However, further research at the molecular and genetic levels is necessary to understand the role of GABA-induced regulations in growth and its associated anatomical mechanisms under salt stress.

Future direction

In present study demonstrates that GABA positively influences the growth of both wheat cultivars under salt stress. Therefore, GABA could be considered a valuable tool for enhancing the quality of agricultural crops, particularly wheat, in salt-affected soils.

Materials and methods

Experimental details

A sand culture experiment was conducted in the wire house of Department of Botany, Division of Science and Technology, University of Education, Township Lahore. Seeds of two wheat cultivars i.e., FSD-08 and ANAJ-17 were obtained from Ayub Agriculture Research Institute (AARI), Faisalabad. Both cultivars were selected based on the wide grown by the local farmers and their variable response to salinity59,60. Both are salt tolerant cultivars being efficient in accumulating Na+ and K+ ions without any adverse effect on the growth to improved yield under salt stress61,62. Seeds were soaked in water overnight, shade dried and subsequently sown in sand filled plastic pots of 22 cm diameter on 30th October, 2020. Each pot was filled with 4 kg of thoroughly washed sand and applied with full strength Hoagland nutrient solution (Epstein, 19). The pots were kept in wire house under natural conditions following average monthly temperature (19 °C), relative humidity (21%) and light intensity (30–52.7 mol/m−2d−1) during the experiment.

Seeds started their germination after 3–4 days of sowing and finalized in almost 8–10 days. Hoagland nutrient solution was applied to the growing seedlings according to the requirement. After 10–12 days of germination, thinning was done to keep 8 seedlings per pot of almost uniform size. Afterward, 21 days old seedlings were salinized with 0 and 120 mM NaCl along with full strength Hoagland nutrients solution. It was given as 50 mM per day to avoid abrupt impacts of salinity on growing seedlings until the desired concentration of 120 mM was obtained at third day whereas the control plants were irrigated with full strength Hoagland nutrients solution only46,63. The GABA was exogenously applied as foliar application with three different concentrations i.e., 1, 2, and 3 mM whereas no-GABA application (0 mM) was taken as control64.

Sampling and data collection

Morphological characters

To determine morphological parameters, total 48 seedlings in each treatment (1 seedling per pot and 12 seedlings from each treatment) were harvested for the measurements of the plant height, root and shoot fresh and dry weight, root and shoot length, number of roots, shoot and leaves. A plastic ruler was used for measuring the seedling length. The root and shoot fresh weight of each plant was weighed by the electronic analytical balance (TM-STE3002S) immediately after harvesting. After weighing, the fresh sample of the root and shoot of each plant was placed in the oven, dried at 65 °C for 72 h to measure the root and shoot dry weight.

Anatomical characters

For anatomical studies, the material was preserved in formalin acetic alcohol (FAA) solution for 48 h. The fixative contained 5% formalin (v/v), 10% acetic acid, 50% ethyl alcohol and 35% distilled water. Plant material was later transferred to acetic alcohol solution (acetic acid 25% and ethyl alcohol 75%) for long-term storage. Permanent free-hand sectioning slides were prepared by serial dehydrations in ethanol (30%, 50%, 70%, 90% and 100%) using standard double-stained technique of safranine and fast green stains65. Photographs were taken by camera fitted with light microscope (Nikon 104, Japan) using an ocular micrometer. Anatomical characteristics related to parenchymatous, mechanical, and vascular tissues were recorded with computerized software (Motic Images plus 2.0).

Statistical analysis

The experimental treatments were arranged in completely randomized design (CRD) with four replications. Analyses of variance of all parameters were performed by the factorial design Procedure of Statistix version 8 (Statistix 8, Analystical, Tallahassee, FL, USA). Comparisons of means among different treatments were made according to the least significant difference (LSD) test at the 5% probability level.