Potassium humate and cobalt enhance peanut tolerance to water stress through regulation of proline, antioxidants, and maintenance of nutrient homeostasis

Water stress is an important factor that substantially impacts crop production. As a result, there is a need for various strategies that can mitigate these negative effects. One such strategy is the application of potassium humate (Kh) and cobalt (Co), which have been reported to enhance the resistance of crop plants. Therefore, the present experiment was designed to investigate whether the application of Kh and Co could positively affect proline, chlorophyll and mineral elements contents, and antioxidant defense systems which in turn will mitigate the negative impact of water stress under different irrigation strategies. In 2021 and 2022, an open-field experiments were conducted by using a split-plot design. The main plots were divided to represent different irrigation strategies (ST), with additional control of full irrigation requirements (ST1). Four STs were implemented, with ST1, followed by the application of 75%, 50%, and 25% irrigation strategies in ST2, ST3, and ST4 respectively, in the next irrigation, followed by the full requirements, and so on. In the subplots, peanut plants were treated with tap water (Control), Kh at 2 g l−1 and 3 g l−1, Co, Co + Kh 2 g l−1 and Co + Kh 3 g l−1. The yield was negatively affected by the implementation of ST4, despite the increase in proline contents. Furthermore, there was a decrease in relative water content, chlorophyll content, antioxidant enzymes, protein, and mineral nutrient elements. However, the application of Kh or Co showed better improvements in most of the studied parameters. It is worth noting that there was an antagonistic relationship between Co and iron/manganese, and the intensity of this relationship was found to depend on the STs implemented. The highest mineral nutrient accumulation, chlorophyll content, relative water content, protein content, oil content, seed yield, and water productivity were observed when peanut plants were treated with Kh 3 g l−1 + Co under the ST2 water strategy.


Experimental design and treatments
As aforementioned in the introduction section, plants are often exposed to various degrees of water stress, which triggers a cascade of several responses.In evolutionary terms, initiated with stomatal closure induced by hormone homeostasis.If water stress is prolonged, the plants tend to modify their morphological traits, which leads to profound negative effects on plant growth, development, and yield 32 .Therefore, to, ensure better growth and align with the research objectives, this study intentionally followed different water stress strategies in which it was in short term and at different intensities to elucidate the response of peanut plants.Hence, the experiment

Crop farming
The recommended fertilizer doses were applied according to 39 as follows: Calcium phosphate (15.5% P 2 O 5 ) was added before sowing at a rate of 480 kg ha −1 .Ammonium nitrate (33.5% N) was added at a rate of 144.0 kg ha −1 divided into 6 equal doses, the first dose was added as a starter dose after 15 DAS, while the remaining doses were added at 22, 30, 40, 50 and 60 DAS.Potassium sulphate (48% K 2 O) was added at a rate of 240 kg ha −1 after 30 DAS.The peanut plants were sown on May 21 and May 20 of 2021 and 2022, respectively at the rate of 180 kg pods ha −1 (120 kg seeds ha −1 ).Two peanut seeds (Giza 6) were drilled per hill and seeded on one side of the dripper's jet after being inoculated with root nodules bacteria (Rhizobium leguminosarum).The seeds of peanut were purchased from the oil crops department, field crops institute and agricultural research center, Egypt.This cultivar is recommended as a high-yielding commercial cultivar.Plants were spaced 30 cm apart in each row, 60 cm between the rows and 5 cm deep in the soil.Moreover, this cultivar and the implemented methods in this study complied with international, national, and institutional guidelines and legislation.

Calculations related to irrigation
Crop evapotranspiration (ETc) The crop evapotranspiration of peanuts (ETc) was determined according to Fao Penman-Monteith 40 , as the following equation: Were, ETc = Crop evapotranspiration (mm).ETo = Reference evapotranspiration (mm).Kc = Crop coefficient.The ST1 (100% of irrigation water requirements) were estimated according to the following equation: were, IR = Irrigation water requirements (mm).ETc = Crop evapotranspiration (mm).Ei = The irrigation system efficiency % (85%) The Ei values were calculated for the 40 cm soil depth based on 41 as average values of 3rd, 7th, 17th and 25th irrigation events using the following equation: where, Ws is the water amounts stored in the root zone (mm) and Wf is the amount of water applied to each plot (mm).Lr = The amount of leaching requirement was calculated according to 42 as: Were: ECw = Electrical conductivity of irrigation water (dS m −1 ).ECe = Estimated electrical conductivity of the saturated soil extract corresponding to a 10% reduction of the maximum yield.
The appropriate timing of the irrigation event and the retention parameters such as (field capacity and wilting point) were determined by the pF-curve (soil water retention curve).Then, the allowed depletion in the available water content was adjusted between 55 and 60%, which was the critical limit for peanut development based on previous studies 43,44 .Accordingly, based on studying the soil moisture behaviour, the soil lost 40% of the available water content after 2 days.Hence, the irrigation event was set every 2 days.The applied water quantities for the other treatments were proportionally obtained from the (100% of irrigation water requirements-ST1) treatment.www.nature.com/scientificreports/Moreover, the mm units were converted to (m 3 ha −1 ) by multiplying with 10, as mentioned by Danielescu et al. 45 .Accordingly, the average total applied water quantities applied to peanut crops during the seasons of 2021 and 2022 were: 4716, 4002, 3582, and 3010 m 3 ha −1 for ST1, ST2, ST3 and ST4 respectively.

The water productivity (WP)
Mathematically (WP) according to Elshamly 46 and can be estimated by: were, WP = The water productivity (kg m −3 ).Y = The obtained yield (kg ha −1 ) and IR = The total irrigation water requirements (m 3 ha −1 ).

Estimation of total chlorophyll, RWC, proline, phenolics, ROS, and antioxidant enzymes in peanut leaves
The total chlorophyll content of the leaves was measured by using a spectrophotometer according to Sadak et al. 47 .The total chlorophyll was estimated in the (100 mg) of fresh peanut leaves which were pulverized and gradually added with 10 ml of the acetone (80%) to extract the total chlorophyll in the dark for 48 h.The extractor was centrifuged at 10,000 × g for 5 min followed by the removal of debris.The absorbance of the supernatant was measured by using a spectrophotometer at 645 and 663 nm for chlorophyll a and b respectively.The average total chlorophyll content was expressed in (%) obtained as follows: The RWC contents of the leaves were measured according to the method of Afzal 48 after 65 DAS.The peanut leaves were collected from ten random plants from each experimental unit.Then the leaves were weighed and transferred to the test tube containing distilled water.The test tube was then kept in the dark at 4 °C overnight until the peanut leaves reached to a constant weight.The dry weight of the leaves was estimated after incubation of the turgid leaves at 70 °C for 24 h.The RWC contents were measured as follows: were, FW: Actual fresh weight of the leaf.DW: Dry weight of the leaf.TW: Turgid weight of the leaf.At 65 DAS, peanut leaves from the top of the plant were randomly collected from ten different plants in each experimental unit.Then, the proline content was estimated using the leaves of the plant as described by Sahin 49 .The 0.5 g of fresh leaf samples were ground in liquid nitrogen with a mortar and pestle.Then 10 ml of 3% sulfosalicylic acid was subsequently added and the solution was then homogenized in a water bath at 99 °C and the solution was stored in the refrigerator until the extraction process.The supernatant was obtained by using a centrifuge at 12,000 × g for 10 min. 2 ml of supernatant was taken and mixed with both (2 ml of glacial acetic acid and 3 ml of 2.5% ninhydrin reagent).For 60 min the reaction mixture was incubated in boiling water, then it was extracted by adding 4 ml of toluene.The absorbance of the proline content was measured using a spectrophotometer at 520 nm and was repeated three times.To determine the proline content the following formula was used: These steps were replicated three times, and the mean value of proline contents was expressed as μ mol g −1 FW.The phenolics content was analyzed according to Abu-Sree et al. 50.Random ten peanut leaves were collected from ten different plants from each experimental unit and dried by using a hot-air oven at 40 ± 3 °C for 36 h.Then crashed and the powder was passed through a sieve mesh cloth.By using aqueous methanol (80%), the leaf powder was extracted and then evaporated by using a rotary evaporator.By mixing 0.2 ml of extracted solution with (1 ml) of the diluted Folin-Ciocalteu's phenol reagent.After that, 0.8 ml sodium carbonate (7.5%) was added.The mixture was left at room temperature for about 30 min and then the absorbance was estimated using a UV spectrophotometer at wavelength 760 nm to measure the total phenolic content, which was expressed as mg gallic acid equivalent (GAE)/100 g of the crude extract against the blank, and the previous steps were replicated three times.
The determination of ROS as hydrogen peroxide (H 2 O 2 ) and the antioxidant enzymes activities such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX) in leaves was estimated according to the method of Dawood et al. 51 and Azevedo Neto et al. 52 .The three replicates for each treatment were used to confirm the results.The peanut leaves were randomly collected from ten different plants from each experimental unit.Then, for the detection of H 2 O 2 content, 0.5 g of peanut leaves were mixed with KH 2 PO 4 -KOH buffer (pH 7.8) to prepare the extract.Then using the TiCl 2, the optical density was measured by using a spectrophotometer at 410 nm and the results were finally expressed in μ mol g −1 FW.
The CAT enzyme activity was determined by preparing a leaf extract from 1 g of fresh peanut leaves with 4 ml of ice-cold extraction buffer consisting of [100 mM phosphate buffer (pH 7.0) + 0.1 mM EDTA + 25 mM H 2 O 2 .].Total CAT enzyme activity was estimated by mixing 100 ml of the supernatant with (2.0 ml) of the reaction mixture consisting of 100 mM phosphate buffer (pH 7.0) + 0.1 mM EDTA + 25 mM H 2 O 2 .The CAT enzyme activity was measured by monitoring the degradation of H 2 O 2 in the range of 1 min at 240 nm, and the results were expressed in μ mol g −1 FW.For the determination of POD enzyme activity, the reaction mixture of Proline content = Toluene (ml) × proline (mg/ml) 115.13  www.nature.com/scientificreports/50 ml was added with 3 ml phosphate buffer (0.1 M, pH 7.0) followed by the addition of 30 ml H 2 O 2 (20 mM) and 50 ml guaiacol (20 mM).Then the mixture was kept in a cuvette for 10 min at room temperature, after that the optical density was estimated at 436 nm and the results were finally expressed in μ mol g −1 FW.
To estimate the SOD enzyme activity in peanut leaves, 1 g of fresh leaves was homogenized with K-phosphate buffer pH 6.8 and 0.1 mm EDTA for 5 min.Then the mixture was filtrated and centrifuged at 16,000 × g for 15 min.After that 50 µl of the enzymatic extract was applied to [13 mm l-methionine + 75 µm nitro blue tetrazolium chloride (NBT) + 100 µm EDTA + 2 µm riboflavin in a 50 mm potassium phosphate buffer pH 7.8].Then the reaction was initiated by turning on the UV lamp and finally, the SOD enzyme activity was measured at a wavelength of 560 nm, and the results were expressed in μ mol g −1 FW.
The APX enzyme activity was measured at 290 nm for 1 min.Using 1.95 ml of the reaction mixture that contained (50 mM phosphate buffer (pH 6.0) + 0.1 mM EDTA + 0.5 mM ascorbate + 1.0 mM H 2 O 2 + 65 ml enzyme extract).The reaction was started by monitoring the degradation of H 2 O 2 in the range of 1 min at 290 nm, and the results were finally expressed in μ mol g −1 FW.

Determination of soil pH
Three soil samples were taken from a depth of 0-40 cm.After that, the soil samples were air-dried and then the soil was passed through a sieve.The mean of the samples was then used to determine the soil pH by a potentiometric method using an Orion 501 digital Ionalyzer research multifunctional pH meter and the soil: water ratio was found to be 2.5:1.

Estimation of mineral nutrient content, carbohydrates, protein and oil content
At harvest, the dried seeds of ten random peanuts from each experimental unit were weighed and ground into a fine powder.The macronutrients such as N, P, K and Ca and micronutrients like Fe, Zn and Mn were estimated according to [53][54][55] .Moreover, the dried leaves and roots of peanuts were weighed and ground into a fine powder to measure K contents using a flame photometer as stated by Estefan et al. 31 .And the previous steps were replicated three times.
The samples of ten different peanut plants (leaves and roots) were collected from Co-treated plots.First, the samples were dried and then crushed into a fine powder.Thereafter, the Co content was determined by using flame atomic absorption spectroscopy in an acetylene-air flame as demonstrated by Wojcieszek and Ruzik 56 .The previous steps were replicated three times, and the mean value of Co contents was expressed as mg kg −1 .The total carbohydrates (mg g −1 ) in the peanut seeds were determined by the method described by El-Katony et al. 57 .The peanut seeds were harvested from ten random plants from each experimental unit, and replicated three times.The 0.5 g of the seeds were ground into a powder and then boiled with 5 ml of 80% ethanol.Thereafter, the mixture was centrifuged for 10 min followed by the extraction.The aliquots were then thoroughly blended with anthrone reagent (8.6 mM anthrone in 80% v/v H 2 SO 4 ).The mixture was then boiled on a water bath at 80 °C for 10 min followed by cooling for 30 min on the ice.Finally, at 623 nm, the absorbance was measured by using a standard calibration curve of glucose.
To determine the protein content and oil content of the peanut seeds, ten seeds were harvested from ten random plants from each experimental unit and replicated three times.The protein content (%) was measured by multiplying the content of nitrogen (N) in seeds (%) with a coefficient of 6.25 by adopting the method of 58 .The oil content of the seeds was estimated by the following formula as described by 59 :

Crop measurements
The peanut crop was harvested at full maturity.The peanut yield was determined for each plot and then converted to Kg ha −1 .

Statistical analysis
Means of variance (ANOVA) were analyzed in all obtained data to determine any statistically significant differences using the CoStat software program CoStat 60 .The means were separated through a revised least significant difference test at the 0.05 level as per Casella 61 .

Ethics approval and consent to participate
This manuscript is an original paper and has not been published in other journals.The authors agreed to keep the copyright rule.

Results
The impact of the various STs, Co and Kh on:

Total chlorophyll
The effects of various STs, Co, and Kh on the total chlorophyll content are presented in (Fig. 1A).There was a significant difference in the total chlorophyll content under various STs as compared to the control treatment as demonstrated in Tables 4 and 5. ST4 led to a decrease in total chlorophyll content under the control treatment.However, the adoption of ST2 and Kh 3 g l −1 applications alone or in combination with Co increased the total chlorophyll content.The highest chlorophyll content was observed under ST2 and the application of Co + Kh

RWC
The results indicated that ST4 under control treatment resulted in significantly lower values for RWC as seen in (Fig. 1B).The maximum increase in RWC was observed when Co + Kh 3 g l −1 was adopted under ST2.

Proline regulation
Proline contents were increased under ST4 and in the first season than the second, as shown in (Fig. 1C).Moreover, the highest values were obtained under the control treatment.In contrast, the lowest proline contents were observed when ST2 was adopted in combination with Co + Kh 3 g l −1 .Relative to control ST2, the proline content of Co + Kh 3 g l −1 under ST2 were decreased in the first and second seasons, respectively.

The activities of antioxidant enzymes (SOD, POD, CAT and APX)
Both Co application and increase water stress intensity led to lower SOD accumulation, as shown in (Fig. 2A).
Under the control treatment in the first and second seasons, SOD content increased by adopting ST1 or ST2 as compared to the ST4.Moreover, supplying Co + Kh 2 g l −1 or Kh 3 g l −1 led to improvements in SOD under various ST schemes as compared to the Co application.Overall, the highest SOD contents were recorded using the Kh 3 g l −1 × ST2.While the lowest SOD content was recorded under the Co × ST4 scheme.As illustrated in (Fig. 2B), adopting the ST2 and the application of Co + Kh 3 g l −1 significantly achieved the highest POD.While the lowest POD contents were recorded under the control treatment of the ST4 scheme in the first season.And under (control ST4, Kh 2 g l −1 × ST4, and Kh 3 g l −1 × ST4) in the second season.On the other hand, the maximum CAT contents were observed by adopting the solitary applications of Kh 3 g l −1 or Kh 3 g l −1 under the ST2 (Fig. 2C).The lowest CAT values were observed with Co application under the ST4 in the first season.And under the control ST4 or control Co × ST4 in the second season.As shown in (Fig. 2D), the ST2 showed the highest APX content under sole or combined applications of Co + Kh.On the other hand, the lowest APX contents were recorded under control ST4 in the first season.And at the ST4 water strategy under sole or combined applications of Co + Kh, in the second season.

H 2 O 2 and phenolic contents
The highest H 2 O 2 level was observed under control ST4 in the first and second seasons (Fig. 3A).Although that was equalled with the adoption of Co + Kh 2 g l −1 × ST4 or the adoptions of Co + Kh 3 g l −1 × ST4.The lowest H 2 O 2 content in the first season was registered under control (ST1 and ST2).However, that was equalled when the sole ) or the combined application of Co + Kh 2 g l −1 under the ST1 scheme was adopted.Likewise, in the second season, the adoptions of control (ST1 and ST2) significantly matched with the sole application of (Co, Kh 2 g l −1 , and Kh 3 g l −1 ) in showing the lowest H 2 O 2 content.However, that was equalled when the combined applications of Co + Kh 2 g l −1 under the ST1 were adopted.The highest phenolic content was obtained with the sole application of Co under the ST4, although that significantly matched the adoption of control ST4 in the first season.While in the second season, the highest phenolic content was obtained with the sole application of Co under the ST4 scheme.On the other hand, the lowest phenolic content was observed when the ST1 was applied in the first and second seasons (Fig. 3B).

Maintaining the nutrient uptake homeostasis of N, P, and Ca
The effects of different STs, Co, and Kh on the uptake of N, phosphorus (P), and calcium (Ca) in peanuts are presented in (Fig. 4A,B and C).The lowest N uptake was observed under ST4 with the sole application of Co.Although that significantly matched the adoption of control ST4 in the first and second seasons.While the highest N uptake was observed under ST1 and ST2 with the combined application of Co + Kh 3 g l −1 .However, that significantly matched the adoption of the sole application of Kh 3 g l −1 under (ST1 and ST2) in the first season.Likewise, the highest N uptake was observed under (ST1 and ST2) with the combined application of Co + Kh 3 g l −1 .However, that significantly matched the adoption of the sole application of Kh 3 g l −1 under ST2 in the second season.
In terms of P uptake, the lowest values were observed by applying combined applications of Co + Kh 2 g l −1 or the sole application of Co under ST4, in the first and second seasons.The highest P uptake was observed with  www.nature.com/scientificreports/ the sole Kh 3 g l −1 applications or both combined applications of Co + Kh 2 g l −1 and Co + Kh 3 g l −1 under ST1 or ST2 in the first and second seasons.The highest Ca uptake was observed with the sole application of Kh 2 g l −1 and Kh 3 g l −1 under ST2.However, that significantly matched the adoption of Co + Kh 2 g l −1 and Co + Kh 3 g l −1 under the ST2 in the first and second seasons.The lowest Ca uptake was observed under the adoption of ST4 with the sole application of Co in the first and second seasons.

Maintaining the nutrient uptake homeostasis of K contents in peanut (leaves, roots, and seeds)
The obtained results indicated that K has a vital role can mitigating the serious repercussions to peanut plants under stressful water conditions by enhancing plant tolerance (Fig. 5A).By comparing the control treatment, it was found that the adoption of various applications had a significant difference in K contents in leaves especially under ST1 or ST2 in both seasons.In the first season, the adoptions of Kh 2 g l −1 under ST2 or Kh 3 g l −1 under (ST1 and ST2) significantly matched with the combined applications of Co + Kh 2 g l −1 and Co + Kh 2 g l −1 under the ST2 by showing the highest K contents in leaves.Likewise in the second season, the adoptions of Kh 3 g l −1 under ST2 significantly matched with the combined applications of Co + Kh 2 g l −1 and Co + Kh 2 g l −1 under the ST2 by showing the highest K contents in leaves.On the other hand, the lowest K content in leaves was observed with the adoptions of the sole application of Co under control ST4 in the first and second seasons.
According to the results presented in (Fig. 5B), it can be observed that the K contents in roots were higher under the extensive irrigation schemes (ST1 and ST2) compared to the (ST3 and ST4) schemes.The highest K contents in roots were obtained when Kh 3 g l −1 was applied with the (ST1 and ST2) schemes.However, that significantly matched the adoption of Kh 2 g l −1 under ST2 or the adoption of Co + Kh 2 g l −1 and Co + Kh 3 g l −1 under ST2 in the first season.Likewise, in the second season highest K contents in roots were obtained by adopting the solitary application of Kh 3 g l −1 under ST1 and ST2.Although that significantly matched the adoption of Kh 2 g l −1 under ST2, or the adoption of Co + Kh 3 g l −1 under ST2 in the second season.On the other side, a significant reduction in K contents in roots was observed in the control treatment where ST4 was adopted or with the adoption of Co + Kh 2 g l −1 and Co + Kh 3 g l −1 under ST4.Regarding the K contents in the seeds (Fig. 5C), the adoption of the ST2 with Kh 2 g l −1 and Kh 3 g l −1 applications matched the combined applications of Co + Kh 2 g l −1 and Co + Kh 3 g l −1 under the ST2 for attaining the highest K uptake in both seasons.On the  www.nature.com/scientificreports/other hand, the lowest K content in seeds in the first season was observed under control ST4 or with the sole application of Kh 2 g l −1 under the ST4.The lowest K content in seeds in the second season was observed under control ST4 under various sole or combined applications.

Maintaining the nutrient uptake homeostasis of Fe, Mn and Zn
It was observed that an increase in water stress intensity leads to a decrease in the accumulation of micronutrients (Fig. 6A).Based on the obtained results in this study, under the control treatment, Fe content increased with the adoption of high irrigation quantities as compared to the low irrigation scheme.The adoption of supplying Co as a sole or combined application led to the lowest values of Fe content in peanut seeds under various ST schemes.However, the application of Kh showed the highest Fe accumulation under the Kh 2 g l −1 × ST2 or Kh 3 g l −1 × ST2, in the first and second seasons.The highest Mn content was obtained with the sole application of Kh 3 g l −1 under ST1, in the first and second seasons (Fig. 6B).Whereas in the first season, the lowest Mn concentration was observed with the sole application of Co under ST4.Although that significantly matched the adoption of the combined applications of Co + Kh 2 g l −1 under the ST4 scheme.In the second season, the lowest Mn content was observed with the sole application of Co under the ST4 scheme.The highest Zn accumulation was noted with the combined application of Co and Kh 3 g l −1 under the ST2 in the first and second seasons.Whereas the lowest Zn content was observed in the control treatment under ST4 in the first and second seasons (Fig. 6C).

Maintaining the nutrient uptake homeostasis of Co contents in peanut (leaves and roots)
In contrast to the full irrigation strategy (ST1), irrigated peanut plants with the stressful irrigation schemes showed the highest accumulations of Co contents in the leaves, as can be seen in (Fig. 7A).Based on the obtained findings, the accumulations of Co contents in the leaves attained lower values in the second season than the first.Moreover, it was observed that the combined applications of Co + Kh 2 g l −1 attained higher Co contents in peanut leaves by adopting ST3 and ST4 in the first season.The same result was observed by adopting ST4 and applying Co + Kh 2 g l −1 or Co + Kh 3 g l −1 in the second season.Conversely, it was shown that the lowest www.nature.com/scientificreports/contents of Co could be attained by applying solitary applications of Co under the ST1 water scheme (0.144 mg kg −1 ), in the first season.In the second season, there were insignificant variations among the solitary Co × ST1 or Co × ST2 in attaining the lowest contents of Co in peanut leaves.On the other hand, the highest Co contents in peanut roots were achieved through the adoption of Co + Kh 3 g l −1 under the ST4 scheme, in the first and second seasons.The lowest Co contents in peanut roots were obtained through the solitary application of Co under ST1 in the first season or by applying Co × (ST1 and ST2) in the second season (Fig. 7B).The highest Co contents in peanut roots were recorded with the combined application of Co + Kh 3 g l −1 under the ST4, in the first and second seasons.

Soil pH and root length
The maximum pH values were achieved through the adoption of ST3 and ST4 in the control treatment, in the first season.While in the second season, the maximum pH values were observed with the sole application of (Kh 2 g l −1 and Kh 3 g l −1 ) under ST3 and ST4 schemes.Although that significantly matched the adoption of control (ST3 and ST 4 ).The lowest pH values were obtained through a combination of Kh 3 g l −1 applications adopting the ST2, in the first and second seasons (Fig. 8A).The highest root length was registered with the combined application of Co with (Kh 2 g l −1 and Kh 3 g l −1 ) under the (ST3 and ST4).However, that significantly matched the adoption of sole Co application under control ST2 in the two seasons (Fig. 8B).Whereas the lowest root length was obtained with the sole application of Co, in the first season, or by adopting either the sole application of Co and the control treatment under the ST4, in the second season.

Oil, protein and carbohydrate contents
Based on the data presented in (Fig. 9A), it can be observed that the oil content in the peanut seeds was higher under the (ST1 and ST2) compared to the (ST3 and ST4).The highest oil content was obtained when a combination of Co + Kh 3 g l −1 was applied with the (ST1 and ST2) schemes.However, that was significantly matched by applying Co × ST2 or Kh 3 g l −1 × ST2 or (Co + Kh 2 g l −1 ) × ST2 scheme, in both seasons.However, a significant reduction in oil content was observed in the control treatment where the ST4 scheme was adopted or when Co was applied under the ST4, in both seasons.Similarly, protein content in peanut seeds was decreased when ST 3 or ST 4 schemes were implemented as depicted in (Fig. 9B).The lowest protein contents were obtained in the control treatment in conjunction with the S4, which was similar to adopting ST4 with the sole application of Co.The highest protein contents in peanut seeds were obtained with the adoption of (Co + Kh 3 g l −1 ) × ST2, in the first and second seasons.
On the other hand, the obtained results indicated that carbohydrate content attained higher increases in the second season than in the first.The maximum increase in the carbohydrate content was observed when a combined application of (Co + Kh 3 g l −1 ) was used along with ST2, in the first and second seasons.While the lowest carbohydrate content was registered with the sole application of Co under the ST4 (Fig. 9C).

Seed yield and WP
The data revealed that peanut production was significantly impacted by the various adopted ST schemes and the examined application treatments, as demonstrated in Tables 4 and 5.The combined application of (Co + Kh 3 g l −1 ) along with the adoption of the ST2 gave the highest value for seed yield, in the first and second seasons (Fig. 10A).While, the adoption of the ST4 along with the Co application resulted in a decreased yield, in the first www.nature.com/scientificreports/and second seasons.Furthermore, the maximum WP was achieved by using Co + Kh 3 g l −1 along with adopting the ST2 and ST3, in both seasons.The minimum WP was obtained by adopting the ST1 in the control treatment, in the first and second seasons (Fig. 10B).

Discussion
The appropriate nutritional and technical irrigation strategies under water stress conditions are considered effective tools to maintain the productivity of field crops to a large extent.

Mechanisms by which plants deal with water stress and the role of Co and Kh under stress condition
A. Proline production Based on the results obtained, it appears that the peanut yield was most negatively affected by the ST4 water stress scheme.The increase in the water stress under the ST4 resulted in the higher production of proline contents.Under stress conditions, peanut plants tend to activate the defense mechanisms such as increasing proline levels.Which perform multiple functions such as osmotic adjustment, maintaining turgor pressure and minimizing the harmful effects of water stress.The previous result aligns with the findings of [62][63][64] .

B. Regulation of proline, antioxidants and ROS
During stress conditions, plants tend to produce a higher amount of ROS which disrupts the metabolic functioning of plants 65 .Therefore, plants tend to accumulate osmolytes such as proline and carbohydrates in the cytoplasm.Where they act as molecular chaperones and help in maintaining protein structure and cell redox status 66 .However, under water scarcity, the water uptake and relative water content decrease leading to an increase in cytoplasmic viscosity.As a result, ROS production continues and causes significant damage,  www.nature.com/scientificreports/particularly in the biosynthesis of chloroplasts and mitochondria 65 .In this study, adopting the ST2 along with Kh alone or in combination with the Co maximally enhanced the antioxidants, which was aligned with the research by 67 .The enhanced content of antioxidants like SOD, POD, CAT and APX is due to the increased level of K + ions and reduces the oxidative stress markers like H 2 O 2 and proline and secondary metabolic compounds (phenolics) content under the water conditions.

Effects of water stress on chlorophyll and RWC alongside the role of optimal ST, Co, and Kh
The reduction in water application resulted in a decreased amount of total chlorophyll content in the peanut plants.The ST2 along with the application of Kh 3 g + Co maximally improved the chlorophyll content.This increase in the chlorophyll content may be attributed to an increased level of K + ions in the leaves which enhances photosynthesis and leads to higher carbohydrate synthesis.Cobalt applications might have improved the chlorophyll contents under various irrigation strategies, which is in line with previous research 17 .The co-application of Kh and Co maximally enhanced the RWC in peanuts under the ST2 water strategy.The www.nature.com/scientificreports/enhancement was attributed to the role of Kh and Co in enhancing the tissue water status and membrane stability index while reducing the electrolyte leakage under water stress conditions.The previous findings corroborate with the results of 13,[67][68][69] .

Effects of Co and Kh under various STon nutrient homeostasis, pH, and root length
The mineral nutrient elements uptake like N, P, K, Ca, Fe, Mn and Zn were highly influenced by the different water strategies along with the application of Kh and Co.The co-application of Kh 3 g l −1 and Co enhances the macro and microelement uptake in plants by improving soil fertility and root growth and development.Moreover, the symbiotic associations with beneficial microbes play a crucial role in plants exploring a large volume of soil and accessing a greater pool of mineral nutrients.The results obtained from this study showed that previous research has highlighted the importance of Co applications for legume crops due to its key role in N-fixation [70][71][72] .However, the application of Co is correlated with the irrigation scheme used 14 .Specifically, the use of the ST4 led to the greatest reduction in yield attributes.This was attributed to changes in soil pH that occurred under stress schemes (ST3 and ST4) that led to a decline in nutrient availability and bio-exudate amounts from both the roots and microbiome in the rhizosphere 17,73,74 .These changes ultimately resulted in increases in soil pH and a decrease in peanut root length.In turn, this has led to a decrease in the availability of both macronutrients (N, P, K, and Ca) and micronutrients (Fe, Mn, and Zn) uptake.Furthermore, the current data indicated the antagonistic relationship between Co and Fe.In a similar context, studies have reported decreases in Fe uptake due to Co applications, although the mechanisms behind this have not yet been clearly defined 10,75,76 .However, Lwalaba et al. 77 have suggested that these reductions are because Fe and Co share the same transporters.Our results confirm these observations and reveal high reductions in Fe accumulation in peanut leaves.The soil pH decreased as the water requirement increased in the soil.The adoption of the ST4 showed the maximum soil pH value.The dearth of water in the soil would have resulted in a greater accumulation of undissolved alkaline salts on the soil surface which would have in turn increased the soil pH.These results are consistent with the previous studies of 78,79 .The increase in water stress would have resulted in a decrease in the root length of the peanut plant.The adoption of the Kh application appeared to confer substantial plant nutrient requirements for root membrane integrity and osmotic adjustment.The Kh also improves lateral root formation and root hair initiation in the test crop plant.Therefore, the Kh applications might have eventually led to improvements in peanut plant tolerance to water stress conditions.Our results are supported by the previous studies of 12,[80][81][82] .However, the combined application of Co and Kh 3 g led to an increase in the root length under the adopted water strategies, especially in the ST2.The increase in the root length might be due to the prominent role of Kh 3 g and Co in regulating root growth and development in plants.These findings are in harmony with those of [83][84][85] .

Hypotheses by which Co increases chlorophyll content and yield under water stress
One interesting finding in the current study was the pronounced improvements of chlorophyll content and yield by applying Co, especially under ST1 and ST2 schemes, although the observed reductions in Fe uptake.Such findings are significant as Fe plays a crucial role in the structure of chlorophyll and its biosynthesis [86][87][88] .This study assumed two hypotheses: (A) that may be attributed to the change in chlorophyll size due to Co molecules.This could be confirmed through microscopic analysis (which we didn't perform in this study).Consequently, this study predicts this modification in the chlorophyll cell size (which is smaller and more numerous).This led to more effective light-harvesting capacity and dry matter production through chlorophyll cells, reflected in improving peanut yield.In this regard 89,90 , demonstrated that smaller chlorophyll cells increase chlorophyll density per unit mass and produce more than 41% of oxygen per chlorophyll unit.While the other hypothesis (B) that absorbed Co molecules interfered with Fe due to their molecular similarity in size and charge 91 .That allowed it to interfere with the related functions of Fe including chloroplast formation.Previous studies, such as those by 92,93 , have reported similar findings for nutrient replacements under certain circumstances.For example, Na + can replace K + in some physiological processes without affecting yield and quality.Liu et al. 79 also indicated that peanut plants showed high absorption activities of Na + to replace the shortage in available K + under salinity conditions.Thus, it was hypothesized that increased Co concentrations in the rhizosphere led to their entrance through either non-selective cation channels 94 or transporters, which caused decreases in Fe uptake.Meanwhile, plants dealt with these amounts of Co by increasing water absorption, as a protected strategy.Hence in this study, notable increases in RWC were observed when Co was applied under different ST treatments.This is supported by previous studies indicating the vital role of Co play in water relations 10,14 and RWC 12 .Nonetheless, some Co molecules interfered with Fe to complete chloroplast formation, as mentioned earlier, which explained the increases in chlorophyll contents.In a similar context, this finding is supported by 95,96 , who noticed a significant favourable impact of Co applications on chlorophyll contents, with the reduction of Fe uptake 10 .They found a strong association between Co and Fe oxides, resulting in the reduction of available Fe for plant uptake.Consequently, some Fe molecules became available within plants, although their total absorbed amounts were lower.At this point, it appears that plants redirected the available amounts of Fe toward increasing the production of antioxidants that Fe consider the main component, such as POD, CAT, and APX 97 .The improvements in POD, CAT and APX were pronounced especially under ST2 when Co was applied.That caused improvements in scavenging ROS components and maintained their concentrations within the safe limits.Therefore, photosynthesis continued, and yield was not affected under these conditions.However, both aforementioned hypotheses were in harmony with the obtained results.Further future studies are required to validate these hypotheses.Since deeper understanding of this issue will provide a basis for developing an appropriate irrigation strategy under Co utilization to improve the yield of peanuts in arid areas.

Optimum treatment combination for peanut crop in arid conditions
The data obtained from the study showed that the combined application of Co and Kh 3 g under the ST2 resulted in the highest oil, protein, carbohydrate and seed yield.Whereas the adoption of the ST2 might provide the requisite amount of water required for the proper physiological functioning of the peanut plants.Moreover, this combination maximally enhanced the chlorophyll content, RWC, antioxidants, mineral elements and root length of the peanut plant.The increases in these parameters would have culminated in enhancing the overall performance of the peanut plants under the different water strategies.This in turn might have led to significant improvements in peanut yield and quality attributes especially under the ST2.
In this scenario, this study hypothesized that under ST2 peanut plants experienced a certain degree of water stress, which triggered a series of physiological effects.This leads peanut plants to rearrange their proline, antioxidant system, and nutrient accumulation to mitigate water stress.Under these circumstances, supplying stressed peanut plants with Co would allow them to improve nutrient uptake and also enhance antioxidant production.Regarding the related mechanisms of improving nutrient accumulation through the application of Co under ST2.Several researchers have noticed similar results; their explanations varied in specifying the decisive interpretation of this result.In this regard, according to the results obtained, the current study contributed mainly for three prominent reasons: A: Co role in enhancing water status within plant tissues by either increasing the uptake of the related nutrient with water relations such as (P, K, Zn, and Ca).The enhancement of K + uptake in peanut leaves resulted in an improvement in the cell membrane permeability and RWC percentage.These results match with the findings of 98,99 .The increase in the uptake of Ca 2+ under these conditions enhances plant cell membrane integrity and improves cellular signalling responses.While P enhances root development and architecture 100,101 .These enhancements in P uptake further contributed to improved carbohydrate synthesis, additionally, it led to raising the nutrient transport due to the decline in the osmotic potential of the sap 44,102 .The enhanced uptake of Zn 2+ contents improves plant cell membrane stability, enhances the formation of chlorophyll, increases the osmolyte accumulation, and stomatal regulation, and increases the rate of photosynthesis 103,104 .B: Activated an effective and equilibrium antioxidant system.That involves the H 2 O 2 and phenolics production, as well as the production of proline and antioxidant enzymes, these findings are in harmony with 105,106 .Where this balance is crucial for maintaining the integrity of cell membranes and alleviating the stress severity of Co and water reductions.Furthermore, that helps peanut plants execute several functions, including enhancing osmotic www.nature.com/scientificreports/potential, preserving turgor pressure, and maintaining the integrity of the cell membranes and proteins 63,107 .C: On one hand, improving peanut root efficiency by enhancing root length and exudates, leading to decreased soil pH and enhancing the availability, acquisition and uptake of P. On the other hand, the application of Kh 3 g l −1 to the stressed peanut plants provided substantial amounts of plant nutrient requirements viz.K + nutrient.This, in turn, has promoted water status and increased root secretions 108,109 by decreasing the soil pH and allowing the nutrient availability and their uptake 81 .Moreover, Kh contributed by the chelating ability in formatting protective layers to maintain the micronutrients, especially Fe and Mn, ultimately causing some improvements in (Fe and Mn).Where the results in this study confirm these observations, these outcomes align with the findings of previous study 110 .Therefore, the current study revealed that the combined application of Co and Kh 3 g l −1 under the ST2 resulted in the highest WP.The enhancement may be due to the improvements in the soil properties and enhancing the antioxidant defense system and the proline contents which has a substantial role in increasing the drought tolerance of the plants.These factors would have in turn enhanced the WP of the peanut plant.

Conclusion
The results indicate that cobalt positively affected chlorophyll contents and peanut yield, despite an observed decrease in iron uptake.This contradicts the role that iron plays in the structure of chlorophyll.The assumption is that any decrease in iron accumulation leads to reduced chlorophyll, especially under stressful water conditions.The study suggested two hypotheses to explain this finding A) The modification in the chlorophyll cells size due to cobalt molecules B) cobalt-iron replacement in the chlorophyll structure.Nonetheless, the current study was unable to confirm these hypotheses due to limitations in the microscopic analysis under the current experimental conditions.Therefore, further research is needed to validate these hypotheses.The study found that the correlation between irrigation patterns and cobalt applications was inconsistent for chlorophyll content, antioxidant enzymes and nutrient accumulation.However, the maximum enhancement was registered in the ST2-adopted water scheme with the application of potassium humate and cobalt.Therefore, based on the findings, it is recommended to implement an ST2 irrigation scheme for the better production of peanuts.In addition, a combined application of cobalt and potassium humate at (3 g l −1 ) is recommended to improve macronutrient and micronutrient accumulation, chlorophyll content, relative water content, protein, oil, yield and water productivity of the peanut plant.This approach can benefit farmers or practitioners by boosting yield and WP of peanuts up to 62.0 and 53%, respectively.Which ultimately achieves the optimum conservation of irrigation and fertilization resources.

Figure 1 .
Figure 1.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) Chlorophyll content, (B) RWC, and (C) Proline contents in the peanut leaves at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 2 .
Figure 2. The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) SOD, (B) POD, (C) CAT, and (D) APX contents in the peanut leaves at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 3 .
Figure 3.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) H 2 O 2 content, and (B) Phenolic contents in the peanut leaves at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 4 .
Figure 4.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) Nitrogen(N), (B) Phosphorus (P), and (C) Calcium (Ca) in the peanut leaves at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 5 .
Figure 5.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the Potassium contents (K) (A) in peanut leaves, (B) in peanut roots, and (C) in peanut seeds at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 6 .
Figure 6.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) Iron (Fe), (B) Manganese (Mn), and (C) Zinc (Zn) in the peanut seeds at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 7 .
Figure 7.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) Cobalt contents (Co) in peanut leaves and (B) Cobalt contents (Co) in peanut roots at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 8 .
Figure 8.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the average (A) Soil pH values, (B) Root length at two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 9 .
Figure 9.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) Oil in seeds, (B) Protein, and (C) Carbohydrates in peanut seeds in two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water).

Figure 10 .
Figure 10.The impacts of implementing different water requirements strategies (STs) and applied cobalt applications combined with ground drench applications of Kh on the (A) Peanut seeds yield and (B) Water productivity (WP) in two seasons of 2021/2022.Data represent the mean of three replicates of both the years of 2021 and 2022.Error bars indicate standard errors from the mean.Bars with different letters are statistically significant at p ≤ 0.05.Abbreviations: Control, spray with pure water; Kh (applying soil application of potassium humate at two rates 2 and 3 g l −1 ); and Co (applied two equal doses of cobalt by (3.25 mg l −1 for the dose), injection in the irrigation water). https://doi.org/10.1038/s41598-023-50714-z

Table 1 .
The monthly meteorological data of the growing seasons of 2021/2022 for the experimental site.The meteorological data were obtained from Toshka Agrometeorological Station, Egypt.Values are the mean of replicates ± standard errors.Max maximum temperature, Min minimum temperature, MS −1 m second −1 , and mm millimeter.

Table 2 .
The physicochemical properties of soil at the experimental site before the cultivation, Egypt in 2021-2022.Each value represents the mean of replications ± standard errors.

Table 3 .
The companied values of the chemical properties irrigation water at the experimental site, during the two growing seasons 2021-2022.Each value represents the mean of replications ± standard errors.TDS total dissolved solids.

Table 5 .
Variance analysis of the investigated parameters in the second growing season.CHL total chlorophyll; RWC relative water content; PROL proline; SOD superoxide dismutase; POD peroxidase; CAT catalase; APX ascorbate peroxidase; H 2 O 2 hydrogen peroxide; PHE phenolics; N nitrogen; P phosphorus; Ca calcium; KL potassium contents in leaves; KR potassium contents in roots; KS potassium contents in seeds; Y peanuts yield; WP water productivity; Fe iron; Mn manganese; Zn zinc; COL cobalt in leaves; COR cobalt in roots; CAR carbohydrates; RL root length; pH power of hydrogen; PROT protein ; NS non-significance.*significance at P ≤ 0.05.