N-ATCA is synthesized from the amino acid L-cysteine and serves as a precursor that is converted back into cysteine in plant. This conversion triggers various metabolic processes, including the synthesis of thiol-rich proteins and the activation of the ascorbate-glutathione (AsA-GSH) cycle, which helps plants manage oxidative stress. N-ATCA undergoes a series of biochemical reactions, resulted in formation of thioproline, formyl cysteine, and L-cysteine.
These compounds play fundamental roles in various biochemical reactions within plants, particularly in the synthesis of glutathione. L-cysteine acts as a precursor for the synthesis of glutathione (GSH), a tripeptide molecule composed of L-cysteine, L-glutamate, and glycine. Glutathione synthesis contributes to protein synthesis by facilitating the formation of disulfide bonds, which are crucial for stabilizing protein structures and maintaining their functionality.
Additionally, L-cysteine is involved in sulfur metabolism, redox regulation, and the biosynthesis of certain phytohormones, such as ethylene and brassinosteroids. Recent studies have also shown its potential to induce disease resistance. The application of N-ATCA on various crops, including New Castle apricot, Starking Delicious apple, Bhagwa Kesar pomegranate, and olive trees. Furthermore, it has been associated with increased fruit set, colour, weight, size, and yield percentage.
Chitosan, a natural polymer derived from chitin, is a valuable substance obtained from the exoskeletons of crustaceans as shrimp, crab, and lobster. Chitosan is produced by removing acetyl groups from chitin, the composition consists of repeating units of glucosamine linked by β-(1,4)-glycosidic bonds. Chitosan possesses numerous advantageous properties, including biocompatibility, low toxicity, non-immunogenicity, and biodegradability, making it highly valuable in various applications.
Chitosan exhibits several beneficial effects on plants, making it a valuable tool in agriculture. These effects include promoting plant growth, increasing tolerance to abiotic stress, improving nutrient uptake and efficiency.
In farming, where conventional practices often lead to significant crop losses due to pests, poor soil, and nutrient deficiencies, nanotechnology offers innovative solutions. Chitosan is a natural biopolymer that has attracted attention in agriculture as a potential ingredient for nanoparticle-based formulations. Chitosan as a nanoparticle primarily acts as a carrier for active ingredients; thus, enhancing their stability, controlling release and improving the bioavailability of active ingredients, than having a direct effect on the physiology of plants themselves.
Reported benefits associated with chitosan-based nanoparticles include improved seed germination, improved long-term and post-harvest quality of produce, improved fertigation, and increased photosynthesis, antioxidant activity, chlorophyll content and tolerance to stress This integration not only overcomes traditional agricultural challenges but also paves the way for more sustainable and efficient agrotechnological systems while maintaining food quality.
Reclaimed semi-arid areas have low organic matter, poor soil structure, limited nutrient availability, and frequent exposure to salinity and drought. These factors generally reduce nutrient uptake efficiency as well as the ability of plants to perform normal functions, resulting in reduced productivity and fruit quality. Thus, the use of biostimulants with a positive environmental impact that can boost nutrient use efficiency and increase the tolerance of plants to stress will be critical to maintaining the production of olives in reclaimed land settings.
The olive tree (Olea europaea L.) plays a pivotal role in the rural economy and lifestyle throughout the Mediterranean region. It is highly prized for the olive oil it produces; a product celebrated for its extensive dietary and medicinal benefits. Olive oil is rich in monounsaturated fats particularly oleic acid, antioxidants, vitamin E, tocopherols, and phenolic compounds, which collectively underpin its importance in the Mediterranean diet.
A key feature of olive oil is its unique fatty acid profile: 70-85% of its unsaturated fats are monounsaturated (primarily oleic acid), supplemented by polyunsaturated fats (mainly linoleic acid), while saturated fatty acids (such as stearic and palmitic acids) constitute around 14% of its composition; the remaining minor compounds account for less than 2%.
The period between late summer and early autumn is crucial for maximizing fruit development and bud preparation for the next season in olive trees. During the late summer through early autumn period, foliar application of chitosan nanoparticles (CHNP) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) can be done to enhance physiological and reproductive functions.
As natural elicitors, CHNP improves the hormone-regulated differentiation of flowers and improves the rate of fruit set, whereas N-ATCA increases the antioxidant capacity of the tree and aids with the metabolism of sulfur to support flower viability and fruit development. Application of each of these products during this period will create the largest impact on fruit production for current season fruit production as well as future flowering, thereby enhancing yield, oil quality, and oxidative stability in semi-arid reclaimed soils.
Vegetative Growth
The results in (Fig. 1A and B) demonstrate that foliar application of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) had significant and multifaceted effects on the growth of Albosana olive trees. (Fig. 1A and B) clearly demonstrates that all treatments positively affected plant growth compared to the control group (T1), and this effect was even more pronounced in the second season, indicating a potential cumulative benefit. The most significant improvement was observed in the combined treatments. Specifically, treatment T16 (1500 ppm CHNPs + 150 ppm N-ATCA) consistently achieved the highest values for shoot length and number of leaves per shoot. T16 increased shoot length by 2.34 ± 0.18 cm and leaf number by 5.6 ± 0.4 compared to the control group.
Leaf nutrients
The results in (Fig. 2A-C) demonstrate that the foliar application of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) had significant and multifaceted effects on the leaf nutrients of Arbosana olive trees. The improved nutritional status shown in Fig. 1 directly supports enhanced growth. Treatment T16 also resulted in the highest accumulation of the main macronutrients nitrogen (N), phosphorus (P), and potassium (K) in leaf tissue (Fig. 4A, B and C respectively). Treatment T16 significantly increased leaf nutrient content compared to the control, with nitrogen (N) at 2.15 ± 0.12%, phosphorus (P) at 0.43 ± 0.03%, and potassium (K) at 1.62 ± 0.08%.
Fruit characteristics
Fruit and oil yield (kg/tree)
As shown in Fig. 3A and B, foliar spraying of CHNPs and N-ATCA significantly improved several fruit parameters in Arbosana olive trees. The data in (Fig. 3A and B) demonstrate that these positive effects directly translate into economic benefits. All treatments increased fruit and oil production per plant, with the combination with the highest CHNP concentration (1500 ppm) showing the greatest effect. Notably, T15 (1500 ppm CHNPs + 100 ppm N-ATCA) achieved the highest yield, even surpassing T16.
This suggests a subtle interaction: the highest N-ATCA concentration (150 ppm) was optimal for vegetative growth (T16), while the slightly lower concentration (100 ppm) favored resource allocation to both fruit and oil production. Treatment T15 significantly increased total fruit production to 18.4 ± 1.2 kg per tree, with oil production also enhanced to 4.6 ± 0.3 kg per tree, resulting in a marked improvement in both fruit and oil yield compared to the control.
Fruit firmness and colour
The data of fruit firmness and colour highlight the significant, multifactorial effects of foliar sprays of CHNPs and N-ATCA on the firmness (Figs. 4) and color (Figs. 5A-C) of Arbosana olive trees. The two treatments differentially modified fruit ripening and quality. Figure 5 shows that fruits treated with CHNPs alone maintained greater firmness, likely due to the film-forming properties of chitosan, which reduces respiration and delays senescence. In contrast, N-ATCA treatment resulted in softer fruits, indicating a faster ripening process.
The color analysis confirms this effect on fruit ripening. The combined treatments, particularly T15 and T16, produced the darkest fruits, characterized by the lowest hue angle (indicating a shift from green to purple/black; Fig. 5A), which is associated with the accumulation of anthocyanin’s and other phenolic compounds, and the highest color saturation (indicating increased color intensity; Fig. 5B). This advanced color characteristic is a key visual indicator of optimal oil accumulation and quality.
Discussion: Synergistic Mechanisms of CHNPs and N-ATCA in Enhancing Olive Growth, Yield, and Ripening
The synergetic use of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) contributed positively to yield and quality through the combination of supportive flowering and fruiting. CHNPs, which are used as natural elicitors in plants, stimulate phytohormone signalling pathways that encourage flower bud differentiation, which increases fruit set. In addition, N-ATCA is a cysteine analog, increases antioxidant activity, and participates in sulphur metabolism, treatment enhancing pollen viability and flower development. The combination of CHNPs and N-ATCA possibly enhances stress tolerance, nutrient translocation, and hormone balance through synergistic effects, leading to increased fruit retention, uniform fruit growth, and optimal oil biosynthesis. Overall, both treatments contribute to vegetative vigour and reproductive performance by achieving increased fruit yield and improved oil quality.
The synergistic effects on vegetative plant growth and nutrient accumulation noted in our studies, especially from the combination of T16 (1,500 ppm-charged hydrogel nanoparticles or CHNPs and 150 ppm of nitrate-amine type carboxylic acid or N-ATCA) appear to have different, yet complementary modes of action that maximize the plants’ ability to conserve their resources for further use. Related chitosan trials on ‘Barhi’ date palm show dose-dependent increases (0.2% chitosan boosted new leaves, length, and area via hormone stimulation).
On an individual basis, we believe the primary function of CHNPs is to assist in the plant’s acquisition and transportation of nutrients. Their small size and positive charge make them good candidates for foliar absorption and cellular entry. Additionally, they may enhance the permeability of cellular membranes and improve the ability of the plant to assimilate and store key macronutrients (nitrogen, phosphorus, and potassium).
Similarly, it appears that N-ATCA is not directly involved in the plant’s acquisition and transportation of nutrients; rather, it prepares the plant for optimum use of those nutrients by priming the internal metabolic environment of the plant. N-ATCA, as a precursor to the amino acids cysteine and glutathione, likely enhances the responsiveness of the plant’s antioxidant system and prevents energy from being diverted away from growth in semi-arid regions due to oxidative stress.
The priming of the internal metabolic environment may also create an environment more conducive for the production of hormones involved in the regulation of cell division and elongation. Therefore, the synergy observed with CHNPs increasing the “aspects” (or supply) of growth-promoting resources and N-ATCA improving the plant’s “capabilities” (or ability to utilize these resources) contributes to the vigour with which vegetative plants have developed in this study over the two growing seasons.
The finding that optimal growth (T16) and highest yield of fruit/oil (T15: 1500 ppm CHNPs + 100 ppm N-ATCA) occurred with different ratios signifies a refined mechanism by which combinations of nutrients interact to affect source-sink relationship or partitioning. The nutrient distribution/manipulation via biostimulants creates a mechanism to adjust the plant’s strategy of resource allocation. Spray Nano-chitosan at 100 ppm for highest yield, and fruit quality in Ewais mango. These nanomaterials enhance nutrient uptake, stress tolerance, and enzyme activity without toxicity risks.
The increased concentration of N-ATCA in T16 likely enhances the metabolic activity and increases the sink strength in the vegetative tissues. In contrast, lower, but still advantageous N-ATCA concentration in T15 and CHNPs appear to support and shift the sink strength towards the reproductive tissues. This is in agreement with previous reports regarding the ability of biostimulants to modulate the relationship between sources and sinks. Improvements in photosynthetic efficiency and nutrient transport via the use of CHNPs may provide the essential photoassimilates and nutrients required to increase the yield, thus eliminating the potential of a trade-off occurring between vegetative and reproductive growth (T15, T16).
The biostimulants had different effects on fruit ripening and had complementary effects on fruit ripeness, which is very important in determining when to harvest and what the oil quality will be. The biostimulant materials CHNPs only delayed fruit ripening by delaying senescence, resulting in fruit that remained firmer longer than un-treated fruits. This increased fruit firmness may have been due to the formation of a semi-permeable film that inhibits respiration and reduces the loss of moisture from the fruit.
N-ATCA stimulated the fruit to ripen sooner by stimulating the accumulation of pigments and producing fruit that became softer sooner probably by influencing the metabolic rates of pig-ment formation and ethylene production. Therefore, the combination treatments (T15, T16) have created a fruit that has optimal skin coloration (a primary indicator of oil accumulation) and does not exhibit excessive softening prior to harvest.
Preharvest foliar sprays of chitosan, nano-chitosan, and calcium chloride on ‘Barhi’ date palms indirectly improved physical properties like fruit firmness and weight loss by reinforcing cell walls and slowing metabolic degradation. The overall improved balance of these reciprocally interacting effects is an important part of managing the best harvest windows for producing oil of the highest quality from this fruit type.
Environmental impact, N-acetylthiazolidine-4-carboxylic acid (N-ATCA) is a cysteine derivative that functions as a plant bioregulator and stress-mitigating compound and is typically applied at very low concentrations. Similarly, chitosan nanospheres are biodegradable materials derived from natural sources and are widely considered environmentally safe biostimulants. Therefore, the combined use of CHNPs and N-ATCA represents a sustainable approach for improving olive productivity and oil quality while reducing reliance on conventional agrochemicals, particularly under semi-arid reclaimed soil conditions.
Conclusion
In conclusion, the integration of chitosan nanoparticles (CHNPs) and N-acetylthiazolidine-4-carboxylic acid (N-ATCA) as foliar biostimulants presents a robust, sustainable strategy for enhancing olive productivity in the challenging conditions of semi-arid reclaimed soils. While CHNPs function as efficient nutrient carriers and protective barriers that enhance foliar uptake, delay senescence, and improve fruit firmness, N-ATCA primes the plant’s internal sulfur metabolism and antioxidant capacity, driving flower viability, fruit development, and accelerated ripening.
Their combined application, particularly at optimized ratios, yields a complementary synergy that not only boosts vegetative growth and nutrient assimilation but also optimally partitions resources between vegetative sinks and reproductive sources. This dual-action approach results in significantly higher fruit and oil yields, improved fruit coloration as a key indicator of phenolic and anthocyanin accumulation, and balanced fruit firmness—critical parameters for high-quality olive oil production.
Embracing Dora’s N-ATCA and Chitosan not only translates scientific breakthroughs into practical farming tools but also empowers growers to enhance yield, elevate oil quality, and build long-term soil and plant health, all while adhering to eco-friendly agricultural practices.