Investigating the effect of chitosanon gene P5CS, PIP, PAL expression in rapeseed (Brassica napus L.) under salt stress

doi:10.21203/rs.3.rs-4912193/v1

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Research Article

Investigating the effect of chitosanon gene P5CS, PIP, PAL expression in rapeseed (Brassica napus L.) under salt stress

https://doi.org/10.21203/rs.3.rs-4912193/v1

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Background

Chitosan, as a non-toxic and biodegradable substance, promotes plant growth and increases the production of secondary metabolites, offering innovative strategies to alleviate plant stress. Salinity is a common abiotic stress that significantly hinders plant growth and development.

Results

This study explores the impact of chitosan on physiological, biochemical, and gene expression responses (PAL, SOD, APX) in salt-stressed Brassica napus L.plants exposed to NaCl concentrations (0, 50, 100, 150 mM). Chitosan application via foliar spray at concentrations of 0, 5, and 10 mg/liter was investigated. The research evaluates gene expression changes (P5CS, PIP, PAL) in Brassica napus L. roots and shoots, highlighting significant alterations induced by chitosan, particularly in PIP expression under saline conditions. Enhanced PAL1 enzyme activity, increased chlorophyll and proline levels, and shifts in iron, potassium, and nitrogen content underscore chitosan's potential to enhance salt stress resilience in plants.

Conclusion

Chitosan application alters gene expression (PAL, SOD, APX), enhances PAL, SOD, APX activity, and boosts chlorophyll and proline levels in salt-stressed Brassica napus L. plants. It also affects nutrient content, indicating its potential to improve plant resilience against salinity, offering valuable insights for agricultural applications.

Chitosan

salinity tolerance

Rapeseed

salt stress damages

oxidative stress

Salinity and other abiotic stressors pose major risks to agriculture and the environment's natural state [1]. Among other abiotic factors, salinity has grown to pose a serious risk [1]. salinity have impacted about one-third of the world's irrigated land, which has a detrimental effect on plant growth [2]. Crop growth and productivity are frequently hampered by salt stress [3]. Specific ion toxicity, nutritional imbalance, and a decrease in the osmotic potential of growing media are the reasons why salt has a deleterious influence on plant growth [2]. The presence of salt stress causes an increase in oxidative damage and reactive oxygen species (ROS), which break down various macromolecules. Thus, the growth and development of the embryo are restricted when α-amylase activity is reduced because it significantly reduces the transfer of sugars [4].

A significant and extensively grown crop all over the world is oilseed rape (Brassica napus L.) Out of oil seed crops grown for human edible oil and animal seed cake, it comes in second place (after soybean) [5]. One common consequence of salinity stress is a decrease in relative water content (RWC) and photosynthesis (dry matter) [6]. The generation of high ROS under salt stress damages the membrane severely, increasing its relative permeability and decreasing its fluidity [7]. Selectivity, flow rate, and ion transport are all impacted, to start with. Yet, it also causes osmotic stress by causing a significant amount of electrolytes to exosmose [8]. MDA, one of the primary byproducts of membrane lipid peroxidation, has the ability to deactivate enzymes and proteins on the membrane, breaking down the biofilm's structure and functionality [9]. An essential indicator of the extent of plasma membrane damage is the MDA content [10].

The breakdown of plant cell structure, the buildup of reactive oxygen species, and the disturbance of ion homeostasis cause the rate of protein synthesis to drop [11]. Since amino acids cannot be synthesised into protein, a buildup of them results in the synthesis of several hazardous compounds [12]. Proline content increased due to salinity stress, primarily in the free amino acid pool in plants and in proline or hydroxyproline content in proteins [13]. The primary cause of soil salinization is the build-up of water-soluble salts in the root zone, such as potassium (K⁺), sodium (Na⁺), chloride (Cl⁻), and sulfate (SO42−) These salts produce osmotic fluctuations, which decrease plant root cells' capacity to absorb water from the soil [14]. Plants' ability to withstand salinity and drought is greatly influenced by plasma membrane intrinsic proteins, or PIPs. PIPs function as essential water channels that control the equilibrium of the water during salt stress [15]. Plant secondary metabolites like flavonoids and lignin have a production pathway that can be opened by Phenylalanine ammonia lyase (PAL) While the plant PAL gene is associated with salinity and alkali, these secondary metabolites are directly linked to the way plants respond to abiotic stress [16]. An enzyme called pyrroline-5 carboxylate synthetase (P5CS) is in charge of initiating the initial stage of proline biosynthesis. One of the two P5CS isoforms, P5CS1 is essential for proline buildup brought on by salt stress [17]. The results of Yang et al. [18]. study, Functional Characterization of the Stipa purpurea P5CS Gene under Drought Stress Conditions, demonstrated that while the deletion mutant sp5cs was less resistant to drought stress, A. thaliana that overexpressed SpP5CS was more resistant than the wild type. Consequently, SpP5CS would be a good choice for a target gene that would increase plant tolerance to drought stress.

This article focuses on the molecular mechanisms behind Brassica napus's responses to salt stress, roles of enzymes (P5CS, PIP, and PAL) and gene expression in Brassica napus's ability to withstand salt by proline, flavonoids, phenol, relative water content (RWC), Malondialdehyde, and electrolyte leakage, as well as chitosan. A thorough understanding of these mechanisms ought to clarify the elements that Brassica napus L needs to develop in order to withstand salt stress conditions.

Plant material

The German hybrid rape seed, variety Rohan, used in this study was purchased from Arman Sabz Company in Adina, Isfahan.

Experimental procedures

Plastic pots were used for the experiment, which was set up in a Complete Randomized Design (CRD) with a factorial arrangement and equal ratios of coquite and perlite mixed in (1:1:1) Four primary irrigation groups were created utilizing NaCl solutions at 0, 50, 100, and 150 mM, each with varying quantities of saline solution. Every prior group was divided into four smaller groups, and rapeseed seeds were treated with varying amounts of chitosan (0.0, 5, 10 mg L − 1) For every treatment, there were three replicates dispersed according to a totally randomized design.

First, for four days, the plants were exposed to 0.05 g/l chitosan. Following that, it received four days of treatment at a 50 mM concentration. They received four days of treatment with 100 mM salt, four days with 150 mM salinity, and four days with 200 mM salinity. Samples were taken at 0, 10, and 20 hours after the treatment concluded.

RNA extraction and the synthesis of cDNA

Lithium chloride extraction was used to obtain total RNA [19–20]. The GeneALL (Cat. NO. 601–602) kit and the procedures outlined in the kit were used to produce the cDNA. First, a 0.2 vial containing 2.5 µl RNA, 0.5 µl dNTP mix, 0.5 µl oligo dT, and 0.5 µl random hexamer was put in the PCR machine and heated to 70°C for five minutes. After that, the vial was taken out of the machine and put in ice for two minutes. After adding 0.5 µl of reverse transcriptase, 0.5 µl of buffer X2, and 0.5 µl of RNasin to each vial, the vial was put in the PCR machine and run at 25°C for 10 minutes, 42°C for 50 minutes, and 80°C for 10 minutes. They remained there for a minute. The produced cDNAs were kept in a refrigerator at -20°C at the conclusion and after.

technique PCR in Real Time

The Real-Time PCR apparatus type ABI (Applied Biosystems Real Time Polymerase Chain Reaction) model StepOne was utilized in this investigation together with the BioFACTTM company's kit. The procedures were followed in accordance with the kit's instructions.

Following the completion of the real-time PCR test and the drawing of the threshold line, the values (Cycle Threshold Ct) obtained from the Phases of the PCR amplification curve were transferred from the gene Rotor software to the Excel environment. The formula below was used to compute the data based on differences in the expression of the Ribosomal 16s RNA gene after sorting in the Gen Ex environment. The SPSS V20 program was used to make graphs and statistical comparisons (Table 1) [21].

formula1: ΔCT = CT (a target gene*) – CT (a reference gene**)

*= PIP, PAL, P5CS

**= mCtrRNA

Calculating Brassica napus.L photosynthetic pigment content

Using Lichtenthaler [22]. approach, the concentrations of chlorophyll a, b, total chlorophyll, and carotenoids (carotene, xanthophyll) in the plant's leaves were measured. Following the pigment extraction process with 80% acetone from 0.1 g of fresh leaves, the leaves were incubated at 4°C in the dark for 48 hours.

Carotenoids (Caro) and other photosynthetic pigments were measured at 663 nm, 647 nm, 550 nm, and 470 nm using a visible spectrophotometer (Cecil BioQuest CE 2501)

The formulae (formula 2, 3, 4,5) below were used to compute the findings.

Formula2: chlorophyll a = 12.25A663.2-2.7A646.8

Formula3: chlorophyll b = 21.50A646.8-5.1A663.2

Formula4: total chlorophyll = chlorophyll a + chlorophyll b

Enzyme extract, PIP, PAL, and P5CS measurements and preparation

Liquid nitrogen was used to compress the plant tissues into a fine powder. 0.5 ml of 50 mM Tris-HCl buffer, pH 7.4, 0.6 M KCl, 7 mM MgCl2, 3 mM EDTA, 1 mM DTT, and PVP (W/V) 5% is insoluble, was combined with 0.25 g of plant tissue to extract each treatment. The plant tissue was then crushed. The homogenized extract underwent filtration using filter paper and was thereafter subjected to centrifugation at a speed of 30,000 g for 35 minutes at 4°C. After that, the supernatant was collected and the P5CS, PIP, and PAL enzymes were measured [22–24]. The reaction mixture consisted of 10 mM ATP, 20 mM MgCl2, 50 mM L-glutamate, and 100 mM hydroxylamine hydrochloride in addition to 50 mM Tris-HCl buffer at pH = 7. Addition of 0.2 ml of enzyme extract resulted in the final volume of 0.5 ml of the reaction solution above.

The reaction was terminated by incorporating 1 mL of stop buffer (2.5 g FeCl3 and 6 g Trichloroacetic acid in a final volume of 100 mL 2.5 M HCl) after the mixture had been incubated for 15 minutes at 37°C. The proteins that had precipitated were centrifuged at 4°C for 15 minutes at 10,000 g, and the optical absorbance was measured at 535 nm. Using the extinction coefficient of 250 M-1cm-1, the quantity of hydroxamate complex generated for the Fe + 3 hydroxamate complex was determined in terms of enzyme units per milligram of protein [25–70].

Enzyme extract preparation and PIP, PAL, and P5CS enzyme activity measurements

The catalytic activity of the enzyme catalase was quantified using the Cakmak and Marschner[26]. (1992) technique. A mixture was prepared by combining 2 milliliters of a 25 millimolar potassium phosphate buffer with a pH of 7, 10 millimolar hydrogen peroxide, and 100 microliters of enzyme extract. The optical absorbance of this mixture was then measured at a wavelength of 240 nm using a spectrophotometer. The quenching coefficient of 40 Mm-1cm-1 was used to compute the quantity of enzyme activity, which was expressed in milligrams of protein. The blank includes 2 ml of 25 mM potassium phosphate buffer at pH 7, including 10 mM hydrogen peroxide [27].

Purification and determination of flavonoids and phenols

Sonald and Lamia's [28] approach was used to determine the content of total phenolic compounds. 5 ml of 95% ethanol was used to pound 0.1 g of the plant's aerial portion, which was then stored in the dark for 24 to 72 hours. After adding 1 ml of 95% ethanol to 1 ml of supernatant solution, the mixture was redistilled with distilled water to reach a volume of 5 ml. Next, 0.5 ml of 50% folin reagent and 1 ml of 5% sodium carbonate were added to the mixture. After an hour of darkness was applied to the mixture, each sample's absorbance was measured at a wavelength of 725 nm. Using the standard curve and the formula (5), the concentration of total phenolic compounds was computed in milligrams per gram of fresh weight. It should be mentioned that sodium carbonate and the Folin Reagent were employed as zero blanks.

Formula 5: y = 0.0042X-0.0836

In order to quantify flavonoids, 0.2 g of plant extract and 3 ml of acidic ethanol (ethanol and acetic acid in a 99:1) were thoroughly combined, and the mixture was centrifuged at 12,000 x g for 15 minutes. Following the straining process, the supernatant solution was heated to 80 degrees and left for ten minutes in a hot water bath. Following cooling, the samples' absorbance was measured using a spectrophotometer at three different wavelengths: 270, 300, and 330 nm [29]; The results were computed using formula (6)

Formula 6: y = 0.0119X-0.029

Enzyme extract preparation and PIP, PAL, and P5CS enzyme activity measurements

The catalytic activity of the enzyme catalase was quantified using the Cakmak and Marschner [26]. technique. A mixture was prepared by combining 2 milliliters of a 25 millimolar potassium phosphate buffer with a pH of 7, 10 millimolar hydrogen peroxide, and 100 microliters of enzyme extract. The optical absorbance of this mixture was then measured at a wavelength of 240 nm using a spectrophotometer. The quenching coefficient of 40 Mm-1cm-1 was used to compute the quantity of enzyme activity, which was expressed in milligrams of protein. The blank includes 2 ml of 25 mM potassium phosphate buffer at pH 7, including 10 mM hydrogen peroxide [27].

Purification and determination of flavonoids and phenols

Formula 5: y = 0.0042X-0.0836

Formula 6: y = 0.0119X-0.029

Extraction and determination of proline

The plant samples were dried and ground into powder in accordance with the procedure nm [30]. 0.1 g of the powder was then added to a mortar along with 10 ml of

3% sulfosalicylic acid, and the mixture was smoothed out using filter paper. A test tube containing two milliliters of the extracted material, 2 ml of ninhydrin reagent, and 2 ml of glacial acetic acid was immersed in a Bain-Marie hot water bath at 100 degrees for an hour. After cooling, each tube received 4 ml of toluene (under the hood), and the tubes were violently shaken for 17–20 seconds to separate the two phases (Toluene is placed at the top due to its lightness and the pink organic phase is placed below) At 520 nm in wavelength, the absorbance of the samples was measured in a spectrophotometer.

Determination of free amino acids

The free amino acid content was measured using the Xiong et al nm [31] approach. Using this approach, 0.5 g of the moist tissue from the aerial section was pounded with 5 ml of 10% ethanol, and its volume was increased to 100 ml using distilled water. Following a clean paper strain, 0.1 ml of the filtered extract was extracted, and 1 ml of acetic acid-sodium acetate buffer, 3 ml of ninhydrin reagent, and 0.1 ml of ascorbic acid were added to it in order to evaluate the free amino acids. For 15 minutes, the final product was heated to 100°C in a bain-marie. Following cooling, the aforesaid solution's volume was raised to 20 ml with 60% ethanol, and a spectrophotometer was used to detect the absorbance at 570 nm. The glycine standard curve was utilized to determine the quantity of free amino acids present in every specimen.

Malondialdehyde (MDA) assay

Heat and Packer's [32] approach was used to test the concentration of malondialdehyde. This procedure involved weighing and rubbing 0.2 g of fresh tissue into a porcelain hollow that held 5 ml of 0.1% trichloroacetic acid (TCA) The created extract was spun in a centrifuge for five minutes at 5000 rpm. Add 4 ml of 20% trichloroacetic acid solution with 0.5% thiobarbituric acid to one milliliter of supernatant solution. After that, the mixture was heated to 95°C in a hot water bath for half an hour. Then, it was promptly cooled with crushed ice. For ten minutes, the cooled mixture was centrifuged at 5,000 rpm. At a wavelength of 523 nm, optical absorbance was measured. A 20% trichloroacetic acid solution with 0.5% thiobarbituric acid was used as a blank for the spectrophotometer. The thiobarbituric-malondialdehyde complex concentration was determined by applying the following formula and the extinction coefficient ε = 155 µMol-1 cm; The results were computed using formula (7)

Formula 7: A = εbc

The absorbance is represented by A, the molar extinction coefficient by ε, the cuvette's path length by b, and the concentration by c.

Quantification of mineral components

The quantity of elements in plants was determined using the Reeves et al [33] approach. Following 48 hours of drying at 70°C in an oven, roughly 0.1 g of each sample's aerial portions were weighed, mashed, and dried for 14 hours at 160°C in an electric furnace. Following cooling, the resultant ash was combined with 3–5 cc of distilled water, filtered, and then the mixture was placed in unique plastic tubes so that the elements' concentration could be measured using an atomic absorption spectrometer.

Relative leaf water content (RWC) measurement

The Wheatherley [34] method was used to measure this index. Two leaves were taken off from each plant, and three 1-cm-diameter discs were made from each leaf. A scale was used to determine weight (FW) After that, the discs were submerged in distilled water for 4 to 5 hours in a Petri Dish plate. The discs were then taken out of the Petri Dish, dried with filter paper, and weighed once again to determine the weight of the entire turgescence state (TW) The discs were covered in aluminum foil, kept in an incubator at 70°C for a whole day, and then weighed to determine their dry weight (DW)

Formula 8 was used to determine the leaves' relative water content:

Formula 8: RWC (%) = ((FW-DW)/(TW-DW)) ×100

FW = fresh weight

DW = dry weight

TW = total turgid weight

Electrolyte leak (EL)measurement

Following the removal of a portion of the plant's fresh tissue, each leaf was cut into 15 discs, which were then prepared and put in closed containers. Then, 10 ml of distilled water was added to them and they were placed at an ambient temperature of 25°C for 24 hours, and after this period, the electrical conductivity of the solution was measured by an electrical conductivity meter (Inob1, Japan) The samples were then placed in a bath of boiling water that was heated to 95 degrees Celsius and left there for fifteen minutes. The samples were removed from the incubator and allowed to cool to room temperature after this time. Simultaneously, the samples' electrical conductivity was assessed once more, and formula 9 was used to determine the permeability of the cell membrane [35].

Formula 9: water leakage= (E1 / E2) ×100

E1 = Electrical conductivity of the solution before boiling

E2 = Electrical conductivity of the solution after boiling

Calculating the percentage and pace at which canola seeds germinate

After choosing 15 healthy seeds and arranging them on filter paper within the plate, each plate received the recommended dosage (5 ml) of chitosan and a solution with varying salinity levels. For eight days, the plates were maintained at a constant temperature of 25 ± 1 degree Celsius in the germinator. The amount and level of the solution in the plates were maintained constant at 2.5 ml during the test period and once every two days. The percentage and rate of germination were calculated using Formula 10and 11 [36].

Formula 10: Germination percentage = ΣG /N ×100

G = number of germinated seeds

N = total number of seeds

Formula 11: Speed of germination = ΣNi /D

Ni = number of germinated seeds

D = number of days

Statistical Analyses

Once the required data was obtained, we ran preliminary statistical tests to adjust the results for analysis of variance (ANOVA) Furthermore, we used Principal Component Analysis (PCA), Heat Maps, and Pearson correlation to evaluate correlations among all the variables. To show a zero mean and a one standard deviation, we first normalized the data because the variables' units of measurement varied. Using SPSS(V20), ClustVis (https://biit.cs.ut.ee/clustvis/), and SRplot (https://www.bioinformatics.com.cn/en), statistical analyses were carried out and values were displayed.

Table 1

A list of the primer sequences utilized in this research
Gen names	Primer names	Primer sequences (forward/reverse)	Product length, nt	Temperature (°C)	NCBI Accession No
18S rRNA	FBRasr	5’-GTATGGTCGCAAGGCTGAAAC-3’	135bp	59.8	KX709367.1
18S rRNA	RBRasrr	5’-GAGCTCTCAGTCTGTCAATCC-3’	135bp	57.8	KX709367.1
PAL11	FPAL11SK	5’- CTTGTTATGGCCTCGAGGATC-3’	128bp	58.2	EU402423.1
PAL11	RPAL11SK	5’- GGCAGCACGAAGGAAACATC-3’	128bp	59.8	EU402423.1
PIP	FHMPIP2	5’-CCGGTATCTCTGGTGGTCAC-3’	128bp	56.9	NM_001316255.1
PIP	RHMPIP2	5’-CCCAACTCCACATATCGCAC-3’	128bp	57.6	NM_001316255.1
P5Cs	FZKPCS	5’-TTACACAAGGTGATCACTGATG-3’	143bp	57.8	XM_009118014.2
P5Cs	RZKPCS	5’-GCTTGTTGCTTCCTCTTGGG-3’	143bp	58.3	XM_009118014.2

Gene expression

Pretreatment of rapeseed with chitosan under saline conditions significantly affected the expression of PIP, P5CS, and PAL in the root section at 5% (P˂0.05) and the shoot at 1% (P˂0.01) PIP gene expression rose in response to the application of chitosan pretreatment, and this rise was sustained over time. Gene expression was same in the plant's two sections in control plants and plants that were taken right away following the most recent treatment. Gene expression alterations were observed in the aerial portion less so than in the root. After the last day of treatment, an upward trend was noticed, with the increase being more pronounced in the aerial portion. At zero time, or the last day of treatment, the expression of the P5CS gene in the aerial and root sections was not significantly different from the control. The PAL1 enzyme activity was enhanced by the impacts of chitosan and salinity; at 100 mM concentration, this increase was substantial, but at 150 mM concentration, there was no discernible change. The application of chitosan had a reducing impact at a concentration of 10 mM, particularly at a dose of 5 mg/liter. The usage of salt and chitosan together increased the activity of the PAL1 enzyme and was statistically significant (P˂0.01) at the 1% level for the phenylalanine ammonia lyase enzyme. The concentration of 10 mg/liter of chitosan with 50- and 100-mM salinity showed the largest increase, whereas the concentration of 10 mg/liter with 150 mM salinity showed the lowest.

Pigment results

Salinity and chitosan had a statistically significant, at the 1% level, impact on the amount of chlorophyll a. With an increase in salinity concentration, the amount of chlorophyll a was lower than in the control group. In rapeseed plants under salt stress, chitosan treatment enhanced the amount of chlorophyll a and chlorophyll b.

Regarding the pigment results of Brassica napus L. plants, we found that in the two groups of untreated and chitosan-treated plants, exposure to salinity stress conditions causes an accumulation of decreasing levels of chlorophyll a and chlorophyll b.

P5CS enzyme activity results

The activity of the enzyme delta-1-proline-5-carboxylate

synthase (P5CS), and Phenylalanine ammonia lyase enzyme (PAL) were found to be significantly higher

(p˂0.01) when treated with chitosan and salt at a concentration of 1%. The comparisons demonstrated the P5CS and PAL enzyme's activity in chitosan- and salt-stressed rapeseed plants. Under salt stress, rapeseed plants treated with chitosan showed enhanced P5CS and PAL enzymes' activity.

Flavonoids and phenols results

The level of flavonoids in rapeseed plants under salt stress has risen due to foliar spraying with chitosan. When chitosan was sprayed foliarly on rapeseed plants under salt stress, the plant's flavonoid content rose. In rapeseed, both the combined and salt and chitosan treatments were statistically significant at the 1% level (P˂0.01) As the concentration of salt grew, the amount of phenol decreased. Moreover, the application of chitosan boosted the synthesis of phenolic compounds, which improved the adverse effects of stress.

Proline results

According to the findings, proline content at level 1 was significantly influenced by chitosan and salt (p˂0.01) Proline levels showed a minor upward trend as salt concentration increased, and a considerable rise in proline was seen when chitosan was applied to canola.

Amino acids results

The findings demonstrated that, at the 1% level, chitosan and salinity both significantly impacted the quantity of free amino acids. The amount of free amino acids in rapeseed increased significantly as a result of using chitosan; this rise was greatest in the Chitosan 10 + NaCl 50 treatment.

The results of Peroxidation of lipids (Malondialdehyde (MDA))

The level of malondialdehyde in the aerial portions of rapeseed increased as a result of salinity stress. Malondialdehyde levels in rapeseed were significantly reduced when chitosan at concentrations of 5 and 10 mg/liter was applied during salt stress(P˂0.05) When chitosan at a concentration of 10 mg/liter was combined with 150 mM salinity, the amount of malondialdehyde was significantly reduced.

Results of evaluation of nutritional elements in rapeseed plant

The results showed that the amount of Fe, Mg²⁺, Ca²⁺, Na⁺, K⁺, Cl⁻, N, and P in the aerial component and root wear significantly affected by salinity, chitosan, and Combination treatment at a 1% level (P˂0.01) As the salt level increased, the amount of iron in the aerial portion significantly decreased in comparison to the control. The amount of iron in the roots of the rapeseed plant has decreased due to salt; the greatest decrease was seen at 150 mM salinity. The amount of iron in the root was not significantly affected by chitosan alone. In comparison to the salt treatment at the same dosage, the combination of

salinity and chitosan

treatment enhanced the amount of iron in the root.

The root portion and the twentieth day following treatment

had the highest salt level, despite the presence of Na⁺ in both the aerial and root parts. The salt content increased in the stem section, with the maximum and lowest amounts being recorded on day 20 and the control, respectively. As compared to the control plants in other groups, there was a lot of Na⁺ buildup in the roots but not as much in the aerial section, therefore chitosan pretreatment had little influence on the Na⁺ accumulation in the roots. Actually, the root kept Na⁺ inside its cells and stopped it from moving to the aerial portion. The root portion had the highest K⁺ concentration when the K⁺ contents of the aerial and root sections were compared. There was a small trend toward a decrease in the K⁺ concentration as the number of days following the treatment's conclusion increased. The rapeseed plant's Cl^- content increased as salt levels rose; this increase was assessed to be 150 mM greater than the baseline level. The level of Cl^- in rapeseed was reduced by using chitosan either by itself or in combination with salt. In rapeseed, salinity reduced N content. The usage of chitosan and salt together was statistically significant(p˂0.01) When chitosan was used, canola plants under salinity stress showed higher N levels than when the salt concentration was the same. Additionally, by treating salinity in conjunction with chitosan, chitosan has been able to make up for the salinity drop. Salinity reduced the phosphorus content of rapeseed. The amount of P in rapeseed increased greater when chitosan alone was used at a dose of 10 mg/liter. The plant's P content rose as a result of the salinity and chitosan treatments combined.

The results of relative water content in rapeseed leaves (RWC)

The relative water content of leaves decreased as salinity concentration increased. The relative water content of leaves increased with chitosan treatment at values of 5 and 10 mg/liter. Chitosan was able to enhance the reduction of the relative water content of leaves that was decreased by salt, according to studies on the interaction impact of salinity and chitosan.

Electrolyte leak (EL) result

The quantity of electrolyte leakage increased with salinity concentration, indicating membrane degradation and decreased membrane stability. The amount of electrolyte leakage decreased after chitosan treatment and was not significantly affected. Researching the interaction between salt and chitosan was statistically significant at the 5% level.

The results of rapeseed germination percentage and speed

The percentage of germination dropped in relation to the control as salinity concentration increased. The germination percentage of rapeseed did not significantly reduce at a concentration of 50 mM, but it did significantly drop at a concentration of 150 mM(P˂0.05) Chitosan showed a significant effect on germination percentage at the level of 1% (P˂0.01) Combining chitosan and salinity as a treatment improved the percentage of germination and lessened the negative effects of salt on seed germination.

Over three, five, and seven days, the germination rate was monitored. In comparison to the control, the germination rate increased over the course of three days at a concentration of 50 mM, but it decreased at a concentration of 100 mM, according to the data. The germination rate was 0% at 150 millimolar concentration. When compared to the control, the germination rate was not significantly affected by the usage of chitosan. The seed germination rate responded favorably to the salt and little amount of chitosan combination treatment. Therefore, it was shown that the germination rate increased at a concentration of 150 mM when combined with chitosan treatment

The result of Principal component analysis (PCA)

The results of principal component analyses of this study are as follows. The biplot's four quadrants' distinction between twelve distinct treatments is displayed by the two-dimensional principal component plot. The plot reveals that the Chitosan 10 + NaCl 50, Chitosan 10 + NaCl 100, Chitosan 5 + NaCl 50, Chitosan 10 + NaCl 150, and Chitosan 5 + NaCl 150, treatments were associated with greater activities of the enzymes PAL, P5Cs, and Gene expression P5Cs and Nutrient k, amino acid, chlorophyll-a, chlorophyll-b, total chlorophyll photosynthetic performance, Flavonoid, MDA, and RWC.

Also, the plot reveals that the NaCl 100, NaCl 50, and Chitosan 5 + NaCl 100 treatments were associated with greater Gene expression PIP, and Nutrient Cl, Na. Interestingly, the strongest association was observed in Pal gene expression when treatments Chitosan 5 + NaCl 100, NaCl 100, and NaCl 50 were applied. It should be mentioned that in treatments NaCl 50 and NaCl 100, the electrolyte leakage exhibited the strongest association. Significantly, the treatments with chitosan 5 and chitosan 100 had the strongest correlations with Mg, P, Ca, Fe, and N, respectively. These treatments also demonstrated a substantial correlation with proline and germination %.

Various approaches Pearson Correlation in Normal, Salinity Stress, and Chitosan Environments

Figure 2 demonstrated the relationships between the different study features. The results showed that the majority of the traits, including enzymes PAL, PIP, P5CS, and gene expression PAL, PIP, P5CS; nutrients (Ca, Mg, P, K, N, Fe, N, Na, Cl), amino acids, chlorophyll-a, chlorophyll-b, and total chlorophyll; proline, electrolyte leak (EL), speed of germination, leaf relative water content

(RWC), phenol, and flavonoids, are correlated with strong positive (dark blue R = 1) and strong negative (dark red R=-1) (Fig. 2).

Plant physiological and biochemical features can be adversely affected by salinity stress, which can lead to a decrease in agricultural output productivity [37]. Abdelkader et al. [38] investigated the expression of the aquaporin gene (Os PIP1-3) in salt-stressed rice (Oryzasativa L.) plants that had received a dopamine pretreatment.The findings indicated that a modest salt treatment (0.15 M NaCl) increased the expression level of OsPIP 1–3. Additionally, it was discovered that the OsPIP1-3 gene contributes to water permeability in rice during salinity stress. Increased resistance to salt and drought stress was demonstrated by transgenic banana plants overexpressing MusaPIP1;2 [39]. There is proof that PIPs play a significant part in the body's reaction to osmotic stressors like salinity [40].

According to Galmés et al [41], in order to preserve the equilibrium between plant water status and water intake, drought-stressed grapevine roots raised the PIP gene's expression level. The delta-1 proline-5 carboxylate synthetase gene enzyme is the most significant enzyme for proline synthesis and plant proline level (P5CS) [42]. Transgenic Arabidopsis with overexpressed LrP5CS genes have improved osmotic, drought, and salt tolerance without adverse effects under unstressed conditions. While under salt stress, lines LrP5CS1 grew better in terms of root elongation while lines LrP5CS2 accumulated less proline than the others. Under conditions of osmotic stress and unstressed growth, the roots of line LrP5CS3 outgrew all others. According to our research, the three LrP5CS genes have different functions in proline accumulation and abiotic stress tolerance, respectively [43].

P5CS1 genes lead to increased proline expression due to salt stress. Subsequently, growth increases and ROS levels decrease [44]. Zhu et al [45] investigated how exogenous Si application affected cucumbers that had been salted. Following Si treatments, they reported an increased ability to withstand salt stress. By regulating Pro at different stress phases through the activities of δ 1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH), as well as P5CS gene expression and interaction with cytokinin, Si administration lowered oxidative stress.

The salinity data in this investigation indicated that there was no significant (P < 0.05) influence of salinity on the relative expression of the PAL1 gene. Gene expression was slightly increased by salinity at concentrations of 50 and 100 mM relative to the control, but non-significantly decreased at 150 mM. This study's modest rise in PAL1 gene expression can be attributed to the following: By altering gene expression, chitosan boosts the synthesis of phenolic compounds and strengthens the plant's resistance to salt stress [46].

Enzymes involved in defense, such as PAL, chitinase, and β-1,3 glucanase, as well as ROS-scavenging enzymes like catalase, peroxidase, and polyphenol oxidase, are all stimulated by chitosan [47].

The results showed that, at 45th DAS, chitosan pretreatment increased PAL activity in sweet basil plants at concentrations between 0.1% and 1%; however, at lower chitosan concentrations of 0.01–0.05%, PAL activity was comparable to control values [48]. These results were consistent with the results of this study. The study conducted by Rashidi et al. in [49] examined THE Effect of chitosan on gene expression, some morphological and physiological traits of sweet basil (Ocimum basilicum L.) under salinity stress. The findings demonstrated that chitosan enhanced growth parameters in both stressed and non-stressed conditions when compared to the controls. In this sense, chitosan boosted the amounts of protein and chlorophyll, as well as the expression of the COVMT and PAL genes, which resulted in a rise in phenolic compounds. These results were consistent with the results of this study.

Plants that are subjected to salt stress might alter their protein composition or produce new proteins as a means of adapting to the changing conditions. Certain metabolites are generated under stress that lessen the harm that salt stress causes. A decrease or rise in structural or enzymatic proteins is the result of variations in the expression of the PAL1 gene, which drives this mechanism [50]. Numerous studies demonstrate the connection between PAL1 and the phenolic pathway, which is evident in plants under abiotic conditions like salt stress [51].

Plant growth phases, biological activities, and chlorophyll content are all impacted by salinity stress.

According to reports, there is a decrease in chlorophyll content when there is salt stress because of increased oxidation and degradation of chlorophyll brought on by the buildup of reactive oxygen species (ROS), and the decrease in chlorophyll is correlated with the salinity level [52–53, 54].

The amount of total, carotenoid, and chlorophyll a, b, and b in the rapeseed plant dropped under salt stress in the current study. As the results demonstrated, there was a significant drop in chlorophyll when salinity stress reached a threshold of 150 ml. Least amount of chlorophyll biosynthesis can also result from glutamate (precursor of chlorophyll and proline synthesis pathway) being used more in the proline production pathway due to competition between glutamine kinase, the proline catalytic enzyme, and glutamate ligase, the first enzyme in the chlorophyll biosynthesis pathway, during salinity stress [55–56]. These results were consistent with our research.

The results showed that chitosan treatment leads to an increase in the amount of chlorophyll a, b and total and carotenoids in rapeseed. The results of Abu-Muriefah's [57] research, the bean plant (Phaseolus vulgaris L.) saw a drop in photosynthetic pigments when subjected to water stress, while this same plant experienced an increase in pigments when chitosan was applied. Furthermore, chitosan is shown to mitigate the negative effects of salt stress in Phaseolus vulgaris L. beans, according to research done by Zayed et al [58] Thus, it has led to a rise in the concentration of carotenoids and chlorophyll a and b. Alenazi et al [37] examined the effects of chitosan (Cs) and chitosan nanoparticles (CsNPs) on Phaseolus vulgaris L. salinity stress. The findings demonstrated that application of Cs or CsNPs significantly improved yield, pigment content, carbohydrate content, proline, and antioxidant system in plants grown under salt stress; additionally, it reduced hydrogen peroxide, lipid peroxidation, and electrolyte leakage. the findings of Jiao et al [59], chitosan controlled the roots, photosynthetic traits, and antioxidant system, which helped maize plants become salinity-tolerant. These findings support the recommendation that CTS be used as a successful strategy to improve corn plants' tolerance to salinity stress. turned into.

Ullah et al [60] looked at the reduction of the negative impact of salt stress on tomato output using exogenous application of chitosan. The findings demonstrated that whilst foliar spraying of chitosan at a rate of 150 mg/L and 150 mM salt stress had a favorable effect on tomato yield, various applications of salinity had a detrimental influence on nearly all parameters evaluated.

Using iron oxide nanoparticles, Moradbeygi et al [61] examined the plant Dracocephalum moldavica L's enzymatic and non-enzymatic antioxidant defense in response to salt stress. The findings demonstrated that the shoots and roots of plants treated with 100 mM NaC1 solution had higher levels of total phenol, flavonoid, and anthocyanin content as well as increased activities of guaiacol peroxidase, ascorbate peroxidase, catalase, and glutathione reductase.

In addition to the present study's findings, a study conducted by Frary et al [62] demonstrated that the Solanum pennelli plant's flavonoid content rose in response to salt stress.

Consistent with this study's findings, another study examined the impact of salinity on the Origanum majorana plant and found that it increased the amount of flavonoids [63]. Researchers looked into how salt affected two types of thyme, Thymus vulgaris L. and Thymus daenensis Celak. They found that under saline conditions, the flavonoid content rose [64]. Consistent with this study's findings, research has demonstrated that rice plants exposed to salt stress have higher total flavonoid content. Flavonoids may have a protective function under stressful circumstances, build up in plant tissue, and shield plants from harm [65].

According to the findings of Coqueiro et al [66], tomato plants' flavonoid content rose when chitosan was used. According to a study on the effects of chitosan under water stress on the white clover plant (Trifolium repens), this plant's flavonoid content increased when it was treated with chitosan and dried out. The accumulation of this compound is thought to be related to the antioxidant defense and osmotic regulation of stressed plants [54]. When thyme (Thymus deanensis Celak) is treated with chitosan, there is a documented rise in flavonoid compounds [67]. The findings on Isatis tinctoria L. and Salvia officinalis L. hair cultures demonstrated that chitosan enhanced the concentration of flavonoid components in these plants [68–69].

In this work, rapeseed plants were grown for 20 days in salty circumstances with chitosan pretreatment increasing the amount of proline in both the aerial and root regions of the plants. Shams Peykani et al [70] shown that coating the seeds with chitosan increased the growth parameters of both seedlings (Triticum aestivum L.; Zea maize L.) and lessened the negative effects of salt. Low quantities of chitosan application resulted in improved proline content and antioxidant enzyme activity as well as decreased MDA buildup. In conclusion, growth performance and biochemical marker variation under salinity stress were improved by chitosan at an adequate dose. According to Sheikha and Al-Maliki [71] and Khan et al [72], the application of varying chitosan concentrations boosts plant defense against various stresses, thereby promoting bean and pea growth and production.

In addition to its function as a useful biomolecule, chitosan is also regarded as a signal molecule and one of the growth regulators [73].

Complete soluble sugars (TSS), proline content, and phenols were shown to significantly increase when chitosan or calcium carbonate was applied to wheat plants, according to a study by Sadak and Talaat [74]

Results on proline in wheat plants under stress from Ku et al [75] concerning Nicotiana benthamiana, El-Bassiouny and Sadak [76] concerning flax plant, and Ibrahim et al [77] concerning sunflower plant (sunflower) verified the rise in proline.

Proline and TSS, the two major components of organic osmolytes, are increasing in plants. This rise may aid in the adjustment of the osmotic potential of cells, resulting in better water absorption and translocation under salinity stress [78].

Proline also functions as a scavenger of free radicals and protects many enzymes and cell structures from oxidative damage [79].

Aazami et al.'s data from 2023 demonstrated that foliar spraying grape plants with CS-SA NCs significantly affected proline levels. The way proline inhibits the growth of ROS, enhances osmotic management, and safeguards membrane structure explains why there has been an improvement in cell protection [80].

One of the most significant roles of amino acids in plants is their ability to withstand salt stress. They accomplish this in a variety of ways, such as by controlling osmotic pressure (proline and glycine are two examples) and eliminating reactive oxygen species [81–82].

Free amino acids can be thought of as the plant's own particular water source while it is under salt stress. They prevent the plant from drying out under pressure by assisting in controlling water intake and preserving the internal water balance of the plant [83]. In this investigation, salt raised the rapeseed's free amino acid content.

Phragmites australis had higher levels of proline and amino acids when exposed to salt, as demonstrated by Xie et al [84] In this study, chitosan at a concentration of 10 mg/liter significantly increased the amount of free amino acids. The amount of free amino acid increased in the other treatments and only decreased at the concentration of 100 mM in the combined treatment of salinity and chitosan.

Rabêlo et al [85] looked into how chitosan affected maize plants that were experiencing water stress. The application of chitosan enhanced the plant's concentration of proline and amino acids, according to the findings.

According to Li et al [54], chitosan application on white clover (Trifolium repens) under water stress resulted in the accumulation of certain amino acids, which is in line with the findings of this investigation. Plants' antioxidant defense system has been activated under stressful situations and osmotic regulation brought about by chitosan's increase in these chemicals.

Active oxygen species are produced by plants under salinity stress, which subsequently leads to oxidative damage. The oxidative stress caused by salt causes membrane damage and lipid peroxidation, with the plasma membrane being the first organ to experience salt stress [86].

The amount of malondialdehyde produced should be evaluated in order to gauge the damage to membranes induced by oxidative stress from salinity [87] Malondialdehyde levels in rapeseed under salt stress have gone up in this study. Taïbi et al [88] found that the bean plant (Phaseolus vulgaris L.) has an increased quantity of malondialdehyde.

In the current study, plants under salt stress saw a decrease in malondialdehyde levels when they were exposed to salinity, and an increase in malondialdehyde in the aerial portions was attributed to chitosan use.

Similar to the current investigation, research by Jabeen and Ahmad [89] demonstrated that low concentrations of chitosan have reduced the amount of malondialdehyde in safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) plants [89]. Turk [90] found in another study that when maize seedlings were subjected to salt stress, the amount of malondialdehyde in the plants increased. However, when chitosan was applied to the plants, the amount of malondialdehyde was greatly reduced and returned to normal levels. Increases in sodium and potassium ions brought on by salinity stress in the soil environment disrupt the natural growth and development of plants [91]. Among these examples are disruptions in the process of water absorption and ion imbalance. Salinity has also caused disruptions in the absorption, transfer, and distribution of nutrients, which upset the ion balance [92]. The findings of this study are in line with the findings of the researchers' investigation. It appears that less potassium is being absorbed as a result of the struggle between potassium and sodium during salinity stress. Potassium ions are accessed by ion channels found in the cell membrane. Because the ionic radii of potassium and sodium are comparable, during salinity stress, sodium enters the cell, causing the amount of potassium to fall and the amount of sodium to increase [93]. The rapeseed plant's concentration of chlorine increased whereas that of phosphorus and nitrogen dropped in the current investigation. According to studies conducted by Wahome et al [94], there has been an increase in chlorine levels, which has disrupted both the cell's metabolism and its ability to absorb components like nitrates.

According to Abdel-Aziz et al [95] findings, adding chitosan to wheat resulted in higher levels of potassium, phosphorus, and nitrogen. According to Rasheed et al [96], chitosan treatment of pea plants (Pisum sativum L.) under cadmium stress resulted in a decrease in the concentration of phosphorus, potassium, and calcium elements.

The notable impact of chitosan on plant development could perhaps be attributed to the heightened activity of nitrogen metabolism enzymes, namely glutamine synthase, reductase, and protease, as well as the enhancement of photosynthesis, which in turn drives plant growth[97–98].

Furthermore, gibberellins and other plant hormones are synthesized in response to chitosan. Furthermore, growth increases several auxins biosynthesis-related signaling pathways via a tryptophanin-independent mechanism [99–76].

Moreover, it could be linked to the elevation of cell osmotic pressure, leading to a higher supply and uptake of water and essential nutrients, as well as the increase in antioxidant and enzyme function, resulting in a reduction of harmful free radicals accumulation [100].

This paper describes the role of chitosan in alleviating salinity stress in Brassica napus L. Chitosan showed a significant impact on the expression of genes, more precisely PIP, P5CS, and PAL genes involved in stress adaptation mainly in segments of roots. It also partly reduced salinity-induced decrease in chlorophyll content by inducing chlorophyll a and b, thus enhancing photosynthetic efficiency under stress. Chitosan treatment enhanced the activities of P5CS and PAL, which improve osmotic stress tolerance by inducing compatible solute and phenolic-compound synthesis. Chitosan foliar spraying increased flavonoids and phenolic compounds, which are key players in free radical scavenging and the reduction of oxidative stress. The increased levels of proline and free amino acids due to chitosan treatments conferred stress tolerance and improved metabolic activity in plants. Chitosan reduced the levels of malondialdehyde in aerial parts and hence improved membrane stability under salt stress. Salinity reduced the uptake of essential nutrients, which was restored by chitosan treatments, hence maintaining nutrient homeostasis and plant health. Chitosan improved leaf RWC, hence attenuating the salt-induced water stress, and reduced electrolyte leakage, hence improving membrane integrity. It strongly improved the percentage and rate of seed germination upon saline conditions. Principal component analysis combined with Pearson correlation analyses demonstrated the strong correlations among enzyme activities, nutrients, pigments, and stress responses across treatments. These results further confirm the role of chitosan in enhancing biochemical pathways to alleviate salt stress in rapeseed, hence promoting sustainable agriculture practices in challenging environments.

Acknowledgements

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The data examined in the present study can be obtained from the corresponding author upon request.

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Competing interests

The authors state that they do not have any conflicts of interest.

Author details

Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran orcid: https://orcid.org/0000-0002-0861-6104.

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Investigating the effect of chitosanon gene P5CS, PIP, PAL expression in rapeseed (Brassica napus L.) under salt stress

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Abstract

Background

Results

Conclusion

Figures

Background

Materials and methods

Plant material

Experimental procedures

RNA extraction and the synthesis of cDNA

technique PCR in Real Time

*= PIP, PAL, P5CS

Enzyme extract, PIP, PAL, and P5CS measurements and preparation

Enzyme extract preparation and PIP, PAL, and P5CS enzyme activity measurements

Purification and determination of flavonoids and phenols

Enzyme extract preparation and PIP, PAL, and P5CS enzyme activity measurements

Purification and determination of flavonoids and phenols

Extraction and determination of proline

Determination of free amino acids

Malondialdehyde (MDA) assay

Quantification of mineral components

Relative leaf water content (RWC) measurement

Electrolyte leak (EL)measurement

Calculating the percentage and pace at which canola seeds germinate

Statistical Analyses

Results

Gene expression

Pigment results

P5CS enzyme activity results

Flavonoids and phenols results

Proline results

Amino acids results

The results of Peroxidation of lipids (Malondialdehyde (MDA))

Results of evaluation of nutritional elements in rapeseed plant

salinity and chitosan

The results of relative water content in rapeseed leaves (RWC)

Electrolyte leak (EL) result

The results of rapeseed germination percentage and speed

The result of Principal component analysis (PCA)

Various approaches Pearson Correlation in Normal, Salinity Stress, and Chitosan Environments

Discussion

Conclusion

Declarations

References

Additional Declarations

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