Proteomic and metabolomic analysis of the Eskimo-1 line on the effect of low positive temperatures

. Low temperatures are a significant stressor that adversely affects plant growth and productivity. They cause mechanical damage to the aerial parts of the plant, while salinity and drought disrupt the ionic and osmotic balance of the cell. Cold damage can also occur due to extremely low temperatures that lead to irreversible damage to plant organs. Consequently, low temperatures are a limiting environmental factor for agricultural productivity. This study aimed to investigate the response of cotton seedlings (lines ESKIMO1 and Coker-312) at the 2-4 true leaf stage to low temperatures. The content of free proline and endogenous salicylic acid was measured after the plants were exposed to temperatures below the critical threshold (+4 0 С, +10 0 С, +15 0 С, and +24 0 С) for 24 and 48 hours in climatic chambers. Analysis of the results revealed changes in the amino acid and phytohormone content after 24 hours of exposure to low temperatures. Based on these findings, we conclude that these substances participate in the adaptation process, protecting plants from the damaging effects of abiotic temperature factors. These results have implications for improving plant growth and productivity under low temperature conditions.


Introduction
The productivity of crops is affected by both regularly and randomly changing environmental conditions. Regular changes, such as seasonal variations, can lead to genetic adaptations in plants [1][2][3][4][5]. However, unpredictable fluctuations, such as extreme cold, drought, or salinity, can significantly reduce crop yield. Thus, it is crucial for scientists to develop new plant varieties that are resistant to these abiotic stresses [4][5][6][7].
Early sowing of crops can be affected by mild and short-term climate changes without causing significant harm to the plants [6]. However, exposure to prolonged and extreme low temperatures during spring in regions with a continental climate can disrupt plant cellular processes and lead to plant death. Such unfavorable conditions can cause critical reductions in physiological functions, including energy metabolism, regulatory systems, and protein metabolism [1,[4][5][6][7].
Initially, it may appear that low temperatures only cause mechanical damage to plants, such as withering, while salinity and drought affect the ionic and osmotic balance of cells, resulting in negative effects. However, positive yet low temperatures can also cause irreversible changes to the structure of plant cells [4][5][6][7][8][9][10]. Low temperatures are a significant factor that limits agricultural productivity. To combat this issue, the Center for Genomics and Bioinformatics of the Academy of Sciences in Uzbekistan is working on creating lines and varieties of cotton that are resistant to abiotic stress, with one such line being the ESKIMO1 [2]. Our research aims to contribute to the global literature on proteomic and metabolomic studies by investigating the mechanisms of resistance to low, positive temperatures.

Materials and methods
The study focused on the transformed cotton line Eskimo-1 (Esk1) and the parental genotype Coker-312 (C-312) of Gossypium hirsutum L. Cotton seeds were planted in pots and placed in a climate-controlled chamber. For the first ten days, the seedlings were grown in 16-hour light and 8-hour dark cycles at a temperature range of 28-32 °C until the first true leaves appeared [4][5][6][7]11]. At the four-leaf stage, the pots were subjected to different temperatures (+4 0 C, +10 0 C, +15 0 C, and +24 0 C) for 48 (2 days) and 120 hours (5 days). Three biological replicates were analyzed for each genomic variant, and 30 plants were used for each replication.
The quantification of Proline (Pro) was carried out using a modified photometric method after staining with acid ninhydrin. The method has a sensitivity of 1 nM and a linear range of 1-100 nMPro [3]. In brief, 1.25 ml of the plant tissue extract was added to 2.5 ml of a 1% solution of ninhydrin in 60% acetic acid containing 20% ethanol. The mixture was placed in 10 ml conical tubes, mixed, covered with glass beads, and incubated in a boiling water bath (95 °С) for 20 min. After cooling, the contents were centrifuged at 10,000 rpm for 5 min, and the optical absorption of the supernatant at 520 nm was determined in 1 cm quartz cuvettes against a sample not containing Pro (empty sample) using a UV 19 spectrophotometer. The Pro content was calculated using the calibration line equation obtained by analyzing known amounts of Pro in the range of 1-20 µg/sample under similar conditions [5][6][7][10][11][12].
To prepare the extraction, 200 mg of finely ground powder of dried cotton leaves were added to 1 ml of extractant consisting of methanol:water:acetic acid=80:19:1 (v/v). The mixture was stirred on a shaker for 2-3 hours, and then centrifuged at 10,000 rpm for 5 minutes. The liquid fraction was separated into a clean test tube, and a second portion of fresh extractant was added to the precipitate [4][5][6][7]10]. The mixture was thoroughly shaken and left for 12-16 hours, then centrifuged again. The supernatants were combined and dried on a vacuum rotary unit. Finally, 0.5 ml of 15% aqueous acetonitrile containing 0.05% acetic acid was added to the dry residue. After shaking on a vibrating mixer, the mixture was centrifuged again and the supernatant was analyzed using the HPLC system described above [4][5][6][7].
The determination of salicylic acid was conducted using high performance liquid chromatography (HPLC) with a Zorbax Eclipse XDB C18 column (3.0x150 mm, 3.5 µm) and a pre-column Zorbax Eclipse XDB C8 (2.1x12.5 mm). Isocratic elution was performed with a mobile phase consisting of 25% acetonitrile (R Chromasolv for LC, Sigma-Aldrich) in 0.05% phosphoric acid at pH 2.5, and the flow rate was set to 0.5 ml/min [8][9][10][11]. Detection was carried out by photometric measurement at 235 nm with a slit width of 4 nm. The column was thermostated at 40°C and had an inlet pressure of 9.5 MPa (95 bar). The sample volume was 20 µl, and the amount of salicylic acid was given per 1 g of dry leaves. A calibration curve was built using five standard solutions of salicylic acid with concentrations ranging from 0.3125-5 μg/ml. The correlation coefficient between peak areas and salicylic acid concentrations exceeded 0.9999. Data processing, calibration curve construction, and result output were performed using Shimadzu Lab Solutions software [4][5][6][7][8].
Statistical analysis was performed using the OriginPro 7.0 software package. The data were analyzed by analysis of variance (ANOVA) and presented as mean ± standard error from three biological and three technical replicates. The significance of differences between mean values was evaluated by the Tukey test, and differences with a p-value less than or equal to 0.05 were considered statistically significant [4][5][6]8].

Results and discussion
The text discusses the observation that during the cotton planting season in spring, there is a sudden decrease in temperature, which can be significant and up to +3 °C. This can cause cold stress in young cotton seedlings, particularly in their cotyledons and first true leaves. The study focuses on the response of the Esk1 line of cotton seedlings to low positive temperatures and how it affects the secondary metabolite proline. The cotyledons and first two true leaves are important in early cotton seedlings as autotrophic organs and are the most vulnerable to cold stress.
The study involved subjecting the Esk1 and C-312 lines to different low-temperature conditions and comparing them to plants grown under normal conditions (+32 °C). After 24 hours of exposure to low temperatures of +4 °C and +10 °C, the seedlings exhibited slight wilting of the leaves. The study aims to understand the effect of low temperatures on the proline content of young cotton seedlings (Fig. 1). The scientific literature reports that proline (Pro) is an important amino acid with several roles in plants, such as regulating the cellular buffer system's redox state. Lehmann and Szabados [4,5] have extensively studied the diverse functions of Pro in plants. The multifaceted and idiosyncratic nature of Pro, along with its synthesis and breakdown processes, explain its significance in plant stress biology [6]. In our research, we investigated the role of Pro in cotton seedlings' response to abiotic stress at an early stage of development when temperature conditions are critical. We conducted experiments using climate chambers and freeze-dried the leaves using a Lyovapor™ L-200 (BUCHI, Switzerland). Pro was extracted and measured using a specific method. The obtained data are presented in Fig. 2.   Fig. 2. Comparison of the content of Pro in the leaves of seedlings of biotechnological and parental lines and hybrids (Eskimo1, Coker-312), which were exposed to low positive temperatures for 48 hours (cold injury). The study investigated the effect of different temperatures on the content of the amino acid proline (Pro) and the stress hormone salicylic acid (SA) in cotton seedlings of two varieties, Esk1 and C-312. The results showed that Pro levels varied in response to temperature changes. At normal conditions (+32 °C), Esk1 had higher Pro content than C-312. However, exposure to lower temperatures resulted in a significant increase in Pro levels in both varieties, with the highest levels observed at +15 °C for Esk1 and +10 °C for C-312. Unexpectedly, at +4 °C, Pro content remained low despite the onset of wilting in the seedlings (Fig. 3).
The study also investigated the concentration of SA in response to temperature stress. Esk1 showed a significant increase in SA concentration at +10 °C and +15 °C, while SA concentration decreased at +4 °C. This pattern was similar to that observed for Pro levels. Overall, the findings suggest that cotton seedlings can adapt to temperature stress by increasing Pro and SA levels. However, the response may vary depending on the variety and the specific temperature conditions. These results have important implications for understanding the mechanisms of plant adaptation to abiotic stress and may inform strategies for improving crop resilience in the face of climate change. Fig. 3. Comparison of the SA content in the leaves of seedlings of biotechnological and parental lines and hybrids (Esk1, C-312, which were exposed to low positive temperatures for 48 hours).
Upon comparison of SA content in leaf tissues of the Eskimo1 line and control genotype, it is apparent that the former exhibits higher accumulation of SA in most experimental variations. An exception to this trend is observed at an incubation temperature of +24 °C for 48 hours, where the SA value of C-312 surpasses that of the Eskimo1 line (21.36±0.09 mg/ml versus 20.2+1.2 mg/ml). Notably, the SA values for the control variant under growth conditions of +32 °C are 20.48±0.05 mg/ml for C-312 and 18.79±0.06 mg/ml for the Eskimo1 line. These observations highlight the potential variation in SA accumulation in response to different experimental conditions and genotypes.
When exposed to unfavorable environmental conditions, plants respond by altering their biochemical processes. These responses can be classified as either adaptive, harmful, accompanying stress, or neutral, depending on the nature and direction of the changes in metabolic processes. To understand how plants respond to prolonged exposure to low  temperatures, we conducted an experiment where seedlings of different genotypes were incubated for 120 hours. The changes in proline concentrations in cotton seedlings under different temperature regimes were observed over time and presented in Fig. 4. The results indicate that the seedlings of the Esk1 line consistently exhibited higher endogenous proline content than the parental genotype across all experimental conditions. Fig. 4. Comparison of the content of Pro in the leaves of seedlings of biotechnological and parental lines (Eskimo1, Coker-312), which were exposed to low positive temperatures for 120 hours.
Similar to the first experiment, we conducted analytical work to determine the accumulation of SA under prolonged exposure (120 hours) to low positive temperatures on the seedlings of the investigated genotypes. The corresponding diagrams can be found in Figure 5. Our findings revealed that under a cold stress of +4 0 C, the SA concentration in Eskimo1 seedlings was 24.9 ± 0.75 mg/ml, while the control had an average of 22.21 ± 0.17 mg/ml. Increasing the temperature stress to +15 0 C resulted in higher SA accumulation, reaching 29.84 ± 0.09 mg/ml. The response of the Eskimo1 line to a temperature stress of +10 0 C was remarkably high, with the highest rate recorded at 55.73 ± 0.19 mg/ml (Fig. 5). When comparing the SA concentrations of the experimental and control genotypes, it was observed that the SA concentration in C-312 was slightly higher (31.11±0.11 mg/ml) than in the Eskimo1 genotype when the seedlings were exposed to a temperature of +15 0 C. This may indicate an adaptation process occurring in plants when they are exposed to changing temperature conditions. The concentrations of Pro and SA presented in the previous experiments suggest that a very low temperature close to the critical temperature of +4 0 C is perceived by seedlings as stressful. The biological processes appear to have been disrupted, as evidenced by the significantly low concentrations of Pro and SA, which were at the same level as the control seedlings. These conditions seem to shock the plants, as there is no difference between high and very low temperatures. It should be noted that the seedlings were initially grown at +32 0 C before being placed in the climatic chambers.
We were interested in studying the response of seedlings to a gradual increase in temperature after being exposed to low temperatures for several days, which is a common occurrence in nature. To investigate this, we conducted an experiment on seedlings with 2-3 true leaves by gradually increasing the temperature from the extreme low of +4⁰С to +15⁰С and +24⁰С over the course of two days. Our data shows that as the temperature increased, the content of Pro in the leaves of the studied lines gradually decreased to a level similar to that of plants grown at higher temperatures. The diagram (Fig. 6) illustrates that the content of Pro in leaves at +4⁰С was 0.699 ± 0.1 µg/g dry weight in the Eskimo1 line. The level of proline decreases to 0.77 ± 0.02 µg/g in seedlings kept at a temperature of 36 ⁰C for 48 hours. On the other hand, seedlings exposed to 32°C for the same duration showed a decrease in Pro to 0.325 µg/g dry wt. When compared to the control genotype, it was observed that the synthesis of the protective amino acid was higher in the Eskimo1 line under extreme conditions. However, at +15 and +24 ⁰C, the Pro content in C-312 plants was lower (0.76 ± 0.04 and 0.62 ± 0.06 μg/g) compared to the Eskimo1 line (1.804 ± 0.07 and 1.33 ± 0.05 μg/g).
Understanding the mechanisms behind a plant's ability to resist the negative effects of cold stress is crucial for agricultural productivity. Previous studies have explored the response of cotton hybrids and transformants to cold injury in combination with other factors, yielding varying results. Temperatures below +15°C have been shown to negatively impact early cotton growth stages. Research has suggested that acclimatization in cotton occurs at temperatures of +20°C, and that resistance to low temperatures is associated with the time of day and light exposure. Additional studies have shown that cotton responds to low temperatures by inducing certain genes. Cold stress and other environmental factors can result in poor germination, chlorosis, leaf growth reduction, wilting, and even tissue death. Cold stress can severely damage the plant's membrane, primarily through acute dehydration during freezing. Cold stress is detected by a receptor on the cell membrane, which then triggers the activation of cold-sensitive genes and transcription factors to enhance stress tolerance.

Conclusions
It was reported that low positive temperatures can have a significant impact on the growth and development of cotton seedlings. Exposure to low temperatures can cause damage to the plasma membrane and tonoplast, resulting in electrolyte leakage and irreversible damage to the plant. However, under stress conditions, plants undergo various metabolic  changes and implement physiological, biochemical, and molecular mechanisms to adapt to the stress. One such mechanism observed in cotton seedlings is the increase in the content of osmoprotectants, such as Pro, which plays a crucial role in stress response.
The results suggest that the use of transgenic approaches can help to increase the resistance of cotton to abiotic stresses, including hypothermia. Overall, the study provides valuable insights into the molecular mechanisms underlying the response of cotton seedlings to low positive temperatures and highlights the potential of biotechnology in developing more resilient crop varieties.