Mitigation of Salt Stress in Camelina sativa by 24-epibrassinolide and/or Salicylic acid Treatments

doi:10.21203/rs.3.rs-3629351/v1

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Mitigation of Salt Stress in Camelina sativa by 24-epibrassinolide and/or Salicylic acid Treatments

https://doi.org/10.21203/rs.3.rs-3629351/v1

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This research was conducted to determine the effects of various hormones EbR and SA applied under salt stress on the growth and development of the camelina plant (Camelina sativa) under greenhouse conditions. Various parameters were evaluated in this study, including FW, DW, PWC, RWC, MP, Ch a, Ch b and carotenoids. The results revealed that salt stress significantly reduced plant growth and productivity of all genotypes in most parameters. The addition of SA and EbR + SA improved the performance of RWC and carotenoid parameters under salt stress, while the addition of EbR increased RWC alone. The highest FW (5.49 g) and DW (1.31 g) were obtained for the NaCl + EbR + SA treatment group after the control group. The highest values of MP were obtained for the NaCl treatment group in Arslanbey genotype and for the NaCl + EbR + SA treatment group for the other two genotypes. Furthermore, the highest increase in Ch a, Ch b and carotenoid contents was obtained for PI-650142 genotype. However, more research is required to fully understand the mechanisms and effects of these compounds when applied together. Overall, SA and EbR have promising potential for improving the productivity of crops grown under salt-stress conditions. Further studies should be performed by considering the sensitivity of genotypes with low salt tolerance.

Camelina sativa

Salt stress 24-epibrassinolide

Salicylic acid

Plant growth and development

Presently, there is a demand for high crop yield to overcome competition in modern agriculture, achieve profit and meet high food demand due to population growth. To achieve high crop yield, agricultural malpractices are being adopted for crop growth, including the application of poor-quality irrigation water. Salinisation, which occurs due to improper agricultural practices, is increasing worldwide in both intensity and extent (Song et al. 2006). In arid regions, the irrigation of the arable land often exacerbates salinisation due to the evaporation of naturally high-salt groundwater (De la Reguera et al. 2020). Therefore, water scarcity and salinity stress following the use of groundwater for irrigation are common in arid and semiarid regions. However, it is estimated that the extent of soil salinisation will increase in the coming years due to climate change (Tester and Langridge 2010).

Salinity stress is one of the most critical abiotic factors that play a role in reducing crop yield and quality. Salinity disrupts the morphology, physiology and biochemical processes of the plant (Vinocur and Altman 2005). The accumulation of Na + and Cl − ions in the tissues of cultivated plants in soils containing high levels of NaCl is one of the most harmful effects of salinity. The high Na + concentration in the soil inhibits the uptake of K+, a major macro-nutrient for plant growth and development. This leads to low agricultural yield and quality and even death of the plant (Wu et al. 2015). Salt stress can decrease photosynthesis by limiting the amount of green pigments (Sudhir and Murthy 2004).

The camelina plant has an effective morphological structure against abiotic stress factors such as high temperature, drought and salinity (Gugel and Falk 2006). The oily and waxy structure of the cuticle layer in camelina prevents water loss from the stomata and thus enables the plant to resist various abiotic stress factors (Razeq et al. 2014). The development of salt-tolerant crops such as camelina is important to ensure the food security of the rapidly increasing world population. The morphological and biochemical analyses performed on camelina plants grown under NaCl concentrations ranging from 50 to 300 mM in vitro to determine their salinity resistance revealed that the plants produced more biomass as a plant defence mechanism against low salinity levels (Morales et al. 2017). Another study showed that the survival probability of camelina is high in salty soil conditions; the relative water content (RWC), plant water content (PWC) and chlorophyll content of the plant decrease under stress conditions, while the amount of membrane permeability (MP) increases (Khalid et al. 2015). Low concentrations (25–50 mM) of NaCl applied at different concentrations during the seedling period of camelina had a positive effect on the fresh weight (FW) and dry weight (DW) of the plant, and the critical salt concentration for seedling growth was 75 mM (Russo and Reggiani 2015). To date, only a few studies have been conducted to determine the morphological and photosynthetic changes that occur in the camelina plant under salinity stress. The identification of species that can grow in saline soils is a good approach to exploit such barren lands.

Many hormones or chemicals, including salicylic acid (SA), melatonin and brassinosteroids (BRs) in particular, are used to increase the resistance of plants against abiotic stressors. However, in terms of low cost and ease of application to large areas, SA is the most frequently used hormone. SA is found in trace amounts in the natural structure of plants, and it is involved in the coordination of physiological processes such as stomatal closure, nutrient intake and protein synthesis (Khan et al. 2003). SA also plays a major role in abiotic and/or biotic stress tolerance and has the potential to exert protective effects on plants under stress conditions (Güneş et al. 2007). Many studies have reported that SA shows an increase in salt-stress resistance in different plant species (Ahmad et al. 2011; Kohli et al. 2018; Zhao et al. 2021).

BRs are another hormonal group that reduces abiotic stress factors and has recently been preferred in applications for stress tolerance. The main goal of BR application to plants under salt stress is to ensure their normal growth. The use of BRs has been one of the most consequential approaches to increase crop production and quality, as they are non-toxic and environment friendly. BRs also function as plant growth regulators that exhibit structural resemblances to animal steroid hormones (Mandava 1988). BRs have the potential to promote tolerance in plants against salinity, drought, high or low temperature and other stress factors (Bajguz and Hayat 2009). Several studies have suggested the curative role of BRs under stressful conditions. BRs increase abiotic stress tolerance by protecting membrane stability (Bajguz 2010; Houimili et al. 2010; Kocova et al. 2010). 24-Epibrassinolide (EbR) is a naturally occurring plant hormone and a type of BR.

Both SA and EbR can induce physiological and biochemical changes that play a critical role in plants during abiotic stress conditions. Because salt stress is considered to have a detrimental effect on plant growth, the present research hypothesised that SA and EbR would mitigate this effect of NaCl stress on the growth and development of camelina. Hence, the present research aimed to investigate whether treatment with SA and/or EbR can decrease the detrimental effects of NaCl stress on camelina and promote its growth.

Plant material and Treatments

In this study, two genotypes (PI-650142 and PI-304269) and one registered variety (Arslanbey) of camelina were tested for salt stress. The plant materials were chosen because of their high seed yield potential and wide adaptability.

The study was conducted using a complete randomised design under controlled greenhouse conditions. Seeds were sowed in pots (25 cm diameter and 20 cm height) filled with a mixture of peat: perlite: soil (1:1:1, v:v:v). There were five plants per pot and five replicates per treatment.

Camelina plants were grown under natural light conditions (day length: 7–8 h), day/night temperatures of 26°C/14°C and relative humidity of 75–80% during the experiment. The plants were irrigated with Hoagland’s solution every 2 days for the experimental duration.

Salt resistance of the camelina genotypes was determined by a preliminary study, and the most effective concentration of 140 mM (salt amount in each treatment: 5.72 g) was selected (Göre 2023). A control group was included to determine the effects of other factors. EbR (0 and 1.5 µM) and SA (0 and 1.5 µM) treatments were performed with Hoagland’s nutrient solution (pH: 7.2; electrical conductivity (EC): 2.2 dS·m^− 1) as the irrigant solution (Hoagland and Arnon, 1950). Table 1 shows the standard composition of Hoagland’s nutrient solution.

Table 1

Composition of Hoagland nutrient solution
Compounds	Concentration of stock solution (mM)	Volume of stock solution of final solution (ml/L)
Macro-nutrients
KNO₃	1000.00	7.00
Ca (NO₃)₂. 4H₂O	1000.00	5.00
KH₂PO₄	1000.00	2.00
MgSO₄. 7H₂O	1000.00	1.00
Micro-nutrients
KCL	25.00	2.00
H₃BO₃	12.50
MnSO₄. H₂O	1.00
ZnSO₄. 7H₂O	1.00
CuSO₄. 5H₂O	0.25
MoO₃	0.25
Fe Na EDTA	64.00	1.00

Irrigation was applied to each pot with 100 mL of irrigation water at 2-day intervals when the plants reached the stage of development of approximately eight leaves at 5 weeks after sowing. NaCl, EbR and SA treatments were applied seven times during the growth period, and the treatments were terminated when the total amount of each treatment was reached (140 mM NaCl, 1.5 µM EbR and 1.5 µM SA). After the completion of the salt treatment, the plants were irrigated at field capacity (200 mL per day) for 10 days.

Table 1.

Plant samples were collected at 50 days from sowing to assess the content of chlorophyll a (Ch a), chlorophyll b (Ch b) and carotenoids in leaves; MP; PWC; and RWC. Plant FW was weighed separately after the plants were dehydrated by placing them on blotting papers for 30 min. Plant DW was obtained by weighing the plants after drying in an oven at 72°C for 48 h (Göre, 2023). Based on the plant FW and DW, PWC was calculated using the following formula:

Plant Water Content\(=\left(\frac{Fresh weight-Dry weight }{Fresh weight}\right)x 100\) (Turner, 1981) (1)

For determining RWC of the leaves, approximately 0.3 g of fresh leaf discs from each plant was weighed, and the wet weight of the discs was recorded. Each leaf sample was individually incubated in a 25 mL beaker filled with water for one night under dark conditions. After completion of the incubation period, each sample was weighed and turgor weight was recorded. After each sample was dried in an oven at 80°C for 24 h, the samples were weighed separately and their DW was recorded. Based on the FW, DW and turgor weight, the RWC of each sample was calculated using the following formula:

Relative Water Content (RWC)\(=\left(\frac{\text{F}\text{r}\text{e}\text{s}\text{h} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}-\text{D}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}{\text{T}\text{u}\text{r}\text{g}\text{o}\text{r} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}-\text{D}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}\right)x 100\) (Gonzalez and Gonzalez-Vilar 2001) (2)

Membrane Permeability (MP)

MP was calculated using the EC meter. Twenty leaf discs of 1 cm diameter each were placed in brown glass bottles. After adding 10 mL of distilled water, the bottles were shaken for 24 h in a shaker at 25°C in a climate chamber at 100 rpm. After 24 h, an EC meter was used to obtain EC1 values. Subsequently, the samples were autoclaved at 120°C for 20 min, and EC2 values were recorded after the samples were cooled to room temperature. Based on the EC1 and EC2 values, MP was calculated using the following formula:

Membrane Permeability = \(\left(\frac{\text{E}\text{C}1}{\text{E}\text{C}2}\right)x 100\) (Lutts et al. 1995) (3)

Chlorophyll-a, Chlorophyll-b and Carotenoids

To determine the content of Ch a, Ch b and carotenoids, 1 cm–diameter leaf discs were taken from a leaf in the middle side of the main shoot of each plant. The leaf discs were transferred to the tubes, and some amount of CaCO3, 3–4 granules of quartz and 4 mL of ethanol (96%) were added to the discs. The leaf discs were crushed to obtain their powder and centrifuged at 10,000 rpm. The solution obtained after centrifugation was subjected to Ch a, Ch b and carotenoid content determination at four wavelengths (710, 665, 649 and 410 nm) by using a spectrophotometer. The content of chlorophyll and carotenoids was calculated according to Visser et al. (2016) by using the absorbance data.

Statistical and data analysis

Analysis of variance of the experimental data was performed using the JMP (2007) statistical software program. Differences in mean values were compared using Tukey’s multiple comparison test (Duncan, 1955).

In addition to genotype (G) and salinity factors, the binary interactions of NaCl×SA and G×NaCl and the ternary interactions of G×NaCl×EbR and G×NaCl×SA were determined to be important for all the evaluated parameters. Among the evaluated parameters, only NaCl×EbR×SA and G×NaCI×EbR×SA interactions for FW; NaCl×EbR, NaCl×EbR×SA and G×NaCI×EbR×SA interactions for RWC; and G×NaCI×EbR×SA interactions for carotenoids were found to be non-significant.

Table 2.

Table 2

Analysis of variance of parameters of three camelina genotypes
Source of variation	df	FW	DW	PWC	RWC	MP	Ch-a	Ch-b	Carotenoids
Genotype (G)	2	329.75**	212.12**	4.29*	215.96**	316.95**	1345.81**	349.40**	1253.56**
Treatment
NaCl	1	11398.42**	789.94**	707.70*	2212.15**	4779.88**	70.24**	17.18**	2741.87**
NaCl*EbR	1	45.14**	1303.70**	511.45**	2.89 ns	1176.60**	2156.53**	572.98**	3029.47**
NaCl*SA	1	780.93**	1500.23**	181.42**	949.12**	36.65**	336.49**	253.28**	1684.49**
NaClEbRSA	1	0.78 ns	12.16 **	7.33**	0.25 ns	46.02**	150.54**	39.73**	15.56**
Interactions
G*NaCl	2	766.93**	741.86**	166.67**	351.18**	430.38**	496.50**	202.23**	2970.24**
GNaClEbR	2	1049.21**	751.72**	417.75**	67.21**	1672.05**	1569.43**	400.60**	1058.87**
GNaClSA	2	151.99**	784.67**	149.14**	496.98**	449.66**	1329.67**	205.91**	1481.58**
GNaClEbR*SA	2	1.26 ns	23.27**	5.86**	0.05 ns	45.48**	143.22**	57.03**	2.57 ns

Moreover, the effect of the binary interaction NaCl×EbR on RWC; the ternary interaction NaCI×EbR×SA on FW and RCW; and the quadruple interaction G×NaCI×EbR×SA on FW, RWC and carotenoid content was non-significant (Table 2). Regarding the genotypes, Arslanbey variety performed better in almost all parameters, except RWC and MP (Table 3).

Table 3

Effects of NaCl, SA and EbR treatments on some parameters of the three camelina genotypes. Mean values followed by the same letter are not significantly different according to Tukey’s multiple range test (P < 0.05)
		FW	DW	PWC	RWC	MP	Ch-a	Ch-b	Carotenoids
Genotype (G)	Arslanbey (G1) PI-304269 (G2) PI-650142 (G3)	8.34 a 8.16 b 6.94 c	1.21 a 1.14 b 1.01 c	86.13 a 85.60 b 86.03 ab	13.40 b 12.64 c 14.11 a	15.14 b 14.62 a 16.54 c	0.74 a 0.50 b 0.43 c	0.30 a 0.17 c 0.23 b	0.89 a 0.21 c 0.63 b
Treatment	Control NaCl NaCl + EbR NaCl + SA NaCl + EbR + SA	12.17 5.37 4.69 5.39 5.49	1.68 a 0.88 d 1.04 c 0.80 e 1.31 b	86.52 b 88.27 a 83.26 d 84.49 c 80.04 e	11.66 16.24 15.46 14.13 13.15	12.27 a 14.55 b 16.47 c 17.94 d 21.64 e	0.90 a 0.46 d 0.58 c 0.48 d 0.61 b	0.36 a 0.20 d 0.24 c 0.24 c 0.30 b	0.81 c 0.64 d 0.80 c 0.88 b 1.14 a
Interactions	G1xcontrol G1x NaCl G1x NaCl + EbR G1x NaCl + SA G1x NaCl + EbR + SA G2xcontrol G2x NaCl G2x NaCl + EbR G2x NaCl + SA G2x NaCl + EbR + SA G3xcontrol G3x NaCl G3x NaCl + EbR G3x NaCl + SA G3x NaCl + EbR + SA	14.18 10.43 6.44 6.61 4.25 15.10 2.30 4.13 4.36 6.95 7.25 3.39 3.50 5.20 5.29	1.72 d 2.01 b 1.45 e 1.00 gh 0.72 i 2.30 a 0.21 k 0.76 i 0.47 j 1.38 e 1.03 g 0.41 j 0.91 h 0.94 ghi 1.83 c	88.86 b 85.81 c 85.74 c 84.98 cd 84.86 cd 85.94 c 93.66 a 78.51 d 84.19 cd 71.18 e 84.77 cd 85.37 c 85.54 c 84.31 cd 84.09 cd	12.58 15.35 13.10 14.76 12.39 9.55 18.20 17.84 12.29 11.66 12.85 15.19 15.44 15.33 15.41	13.39 gh 17.15 de 16.46 ef 17.02 def 16.26 f 10.82 i 8.71 j 19.61 b 14.15 g 30.25 a 12.61 h 17.82 cd 17.76 cd 18.26 c 18.44 c	1.72 a 0.48 f 0.74 c 0.74 c 1.08 b 0.63 d 0.56 e 0.34 g 0.35 g 0.21 h 0.36 g 0.36 g 0.66 d 0.37 g 0.56 e	0.72 a 0.23 e 0.34 c 0.28 d 0.40 b 0.25 de 0.19 f 0.12 g 0.14 g 0.08 h 0.13 g 0.17 fg 0.25 de 0.30 cd 0.42 b	1.04 0.65 1.01 1.00 1.53 0.74 0.70 0.45 0.58 0.36 0.65 0.55 0.83 1.05 1.55
Q_{0.05 G}:2.381; Q_0.05 Control: 4.893; Q_{0.05 NaCl}:2.908; Q_{0.05 NaClxEbR}:2.615; Q_{0.05 NaClxSA}:2.615; Q_{0.05 NaClxEbRxSA}:3.099; Q_{0.05 GxNaClx EbRxSA}:3.745

Table 3.

FW and DW

A decrease in FW and an increase in DW in a salinity-stressed plant indicate that the plant is undergoing osmotic adjustment, a process that retains water in response to salt stress. The plant attempts to counteract the effect of salt by maintaining a balance of water and ions within its cells. A comparison of the FW and DW of the plant reveals the amount of water lost by the plant because of salt stress. The loss of a significant amount of water can negatively affect the plant’s growth and total plant biomass.

The FW of all genotypes decreased due to salinity stress. However, EbR, SA and EbR + SA interaction treatments improved FW in PI-650142 and PI-304269 genotypes (Fig. 1a). Similar results were reported for SA treatment in different plants (Kaydan et al. 2007; Noreeen and Ashraf 2008).

The highest values of FW and DW (8.34 and 1.21 g, respectively) were obtained for Arslanbey variety (Table 3). The highest FW was obtained for the NaCl + EbR + SA (5.49 g) treatment group as compared to the control group, although the difference was not statistically significant. The DW parameter showed results similar to those of the FW parameter, with a statistically significant difference; the highest DW was obtained for the combined treatment (NaCl + EbR + SA) group (1.31 g) as compared to the control group. The genotypes differed in their sensitivity to abiotic stress factors. Compared to the control plants, Arslanbey, PI-304269 and PI-650142 showed a significant decrease in FW under NaCl stress (26.44%, 84.76% and 53.24%, respectively). Following the treatment with EbR and/or SA, the FW again increased except for that of Arslanbey variety. The NaCl + EbR + SA treatment led to a higher DW (1.83 g) only for PI-650142 genotype as compared to that for the control group (Fig. 1b). Furthermore, compared to the control group, Arslanbey variety showed the highest DW under NaCl (2.01 g) treatment. For the genotypes other than Arslanbey, single or combined treatment of hormones (SA or EbR) removed the reducing effect of salt on DW.

Several studies have reported that EbR treatment can improve the FW and DW of different plants under saline conditions, although similar research on the camelina plant is yet to be reported (Shahbaz et al. 2008; El-Khallal et al. 2009; Shahid et al. 2011). A previous study conducted on canola, a close relative of camelina, showed that the growth of canola was remarkably inhibited by 150 mM NaCl, and this adverse effect was significantly alleviated by foliar spraying with EbR (Liu et al. 2013). Similar results were noted for Brassica juncea treated with 24-EbR or 28-HBL (Homo-Brassinolide) (Hayat et al. 2007; Ali et al. 2008).

Figure 1.

The high resistance against salt stress in plants might be due to the coadministration of SA and EbR, which complement the activity of each other through different mechanisms. The combination of these two hormones may lead to a higher potency, thereby resulting in higher FW and DW of the plants in the current study. This suggests that the combination of these two hormones provides a more comprehensive solution to the negative effects of salt stress on plant growth and development.

PWC

Arslanbey variety showed the highest PWC value (86.13%) among the genotypes, while the NaCl treatment showed the highest PWC value (88.27%) among the treatments (Table 3). The highest PWC values for each genotype under different treatments were also estimated. Arslanbey genotype showed the highest value (88.86%) in the control group, PI-304269 genotype showed the highest value (93.66%) in the salt treatment group and PI-650142 genotype showed the highest value (85.54%) in the NaCl + EbR treatment group. All genotypes showed the lowest PWC values for the NaCl + EbR + SA treatment group (Fig. 2a). The greatest change in the PWC value (24%) occurred in the PI-304269 genotype (from 93.66–71.18%).

PWC provides vital information regarding the plant’s hydration status, growth conditions and overall health. PWC is affected by several factors, including plant genotype and growth conditions. High PWC values generally indicate that the plant is well hydrated and in good health, while low PWC values represent dehydration or stress due to environmental factors such as high temperature, drought or salinity. PWC also provides information regarding the plant’s ability to uptake and utilise water, which is essential for growth and survival. A high PWC value indicates optimal conditions for growth and development, while a low PWC value shows that the plant is experiencing stress in the process of tolerating adverse environmental conditions or other factors.

Salt treatment can increase PWC due to osmotic potential (Zaki and Radwan 2021). When salt is applied to soil, it creates an osmotic pressure that draws water from the roots to the shoots, leading to an increase in the water content of plant tissues. Conversely, in the absence of salt application, the osmotic potential is low, thereby causing the plant to lose more water and leading to lower water content in the plant.

The reason that plants treated with salt and SA have higher water content than those treated with salt and EbR could be the different effects of these compounds on the plant’s water balance. SA is a naturally occurring hormone in plants, and it is involved in various stress responses, including osmotic stress. SA can increase plant tolerance to salinity by increasing water uptake and reducing water loss. EbR, alternatively, is a plant growth regulator that promotes growth and development. The effects of EbR on plant growth and water balance probably differ from those of SA, thereby leading to a lower water content in plants treated with salt and EbR than in those treated with salt and SA. Further research is necessary to confirm these findings and identify the underlying mechanisms.

RWC

RWC indicates the plant’s hydration status relative to its maximum capacity for water uptake. It is used to monitor changes in the plant’s hydration status over time and to evaluate the effect of environmental factors on plant health. RWC can also be used to diagnose the causes of plant stress and as an indicator of plant health. High RWC values indicate that the plant is well hydrated, while low RWC values show that the plant is experiencing stress due to dehydration or other environmental factors.

The highest RWC value (14.11%) was detected in the PI-650142 genotype. The lowest RWC value (12.64%) was noted in the PI-304269 genotype. Thus, the susceptibility of PI-304269 genotype to stress is higher than that of PI-650142 genotype.

A multiple comparison test for the effects of different treatments on RWC could not be performed due to the lack of significant difference among the treatments (Table 3). For NaCl treatment, Arslanbey and PI-304269 genotypes showed the highest RWC value of 15.35% and 18.20%, respectively. PI-650142 genotype showed the highest RWC value (15.44%) for NaCl + EbR treatment (Fig. 2b).

Treatment of the plants with SA or EbR in the presence of NaCl improved the RWC of the plants. Agarwal et al. (2005) showed that the treatment of wheat with SA increased its RWC capacity. Previous studies on different plant species have also indicated that EbR and/or SA could enhance the water retention capacity by promoting water uptake and increasing RWC (Karlidag et al. 2011; Mohammadreza et al. 2012; Kohli et al. 2018). This finding suggests that EbRs may also contribute to an increase in RWC capacity, potentially through their positive effects on water uptake and retention.

The exposure of plants to high levels of salt tends to decrease their RWC (Karimi et al. 2005). This reduction in RWC could indicate diminished turgor pressure, which can subsequently limit the availability of water for the cellular expansion process.

Plants exposed to high-salt concentrations may experience reduced turgidity in their leaves. However, this effect can be mitigated by treatment with EbR and SA. These compounds help to retain water within the leaves and increase solute accumulation in the cytosol, thereby preventing further loss of turgidity. Ultimately, this enhances the plant’s ability to cope with salt stress and maintain healthy growth.

MP

MP refers to the loss of cellular content from a plant cell because of cell membrane damage. MP can occur due to various biotic and abiotic stress factors such as diseases, physical damage, high temperature and salinity stress. Stress factors could drastically affect the permeability of the plant cell membrane, leading to decreased growth, decreased yield and even plant death. Hence, it is critical to measure MP to assess the stress status of the plant and to determine the severity of the effects of environmental factors on plant health. A high level of MP indicates that the plant is experiencing stress, while a low level of MP shows the normal course of the vital activities of the plant.

NaCl treatment significantly increased MP as compared to the control group. PI-304269 showed the lowest MP value (14.62%) among the genotypes. Among the treatments, the control group (12.27%) showed the lowest MP value, followed by the NaCl treatment group (14.55%). For the combinations of genotypes and treatments, the control group showed the lowest MP value in all genotypes, except PI-304269. The highest MP values were obtained for the NaCl treatment group for Arslanbey genotype and in the NaCl + EbR + SA treatment group for the other two genotypes (Fig. 2c). Furthermore, the lowest MP value (3.76%) was shown by Arslanbey variety, while the highest value (5.83%) was shown by PI-650142 genotype. For PI-304269 genotype, the difference in MP values between treatments was 21.54%. In this genotype, the NaCl + EbR + SA treatment increased the MP value (30.25) by approximately threefold as compared to that of the control group (10.82). This further confirmed that PI-304269 genotype is highly affected by salinity and is less responsive to hormone treatment.

Figure 2.

Ben-Ahmed et al. (2009) demonstrated that the addition of SA to the growth medium of tomato plants grown under salt stress considerably reduced the harmful effects of stress by maintaining MP (as measured by ion permeability) and photosynthetic pigments. Another study reported that MP increased as salinity stress increased in rapeseed plants but decreased when 1 mM SA was applied to these plants (Mohammadreza et al. 2012). Similarly, Stevens et al. (2006) showed that the treatment of tomato with 150–200 mM NaCl and 0.1 mM SA increased the MP value of the plant. In general, MP can provide critical information about the overall health of a plant and its ability to survive and sustain under different environmental conditions. This information can be useful to understand the impact of environmental factors on plant growth and survival and to develop strategies to protect plants from stress. BRs modify the structure and stability of the plant cell membrane under stress conditions (Hamada 1986). Thus, in the present study, EbR-treated plants under stress showed higher MP values.

Chlorophyll-a, chlorophyll-b and carotenoid

Ch a, Ch b and carotenoids are three types of pigments found in plants, and they play important roles in photosynthesis. The contents of Ch a, Ch b and carotenoids in a plant can reflect its photosynthetic ability, overall health and response to environmental stress. For example, a decrease in the contents of Ch a or Ch b can indicate that the plant is experiencing stress or disease, while an increase in the content of carotenoids can indicate that the plant is responding to environmental stress or high light levels. In general, estimating the contents of Ch a, Ch b and carotenoids in a plant can provide critical information regarding the plant’s ability to conduct photosynthesis and respond to environmental stress. This information can be useful to monitor plant growth and health and to develop strategies to protect plants from environmental stress and diseases.

The increase in the chlorophyll content of camelina plants treated with salt is probably due to salt stress–induced changes in the physiology and metabolism of the plant. Salt stress can stimulate the plant’s defence mechanisms, leading to the increased production of antioxidants and other protective compounds, including chlorophyll. Salt can also stimulate the activity of enzymes involved in chlorophyll synthesis, thereby further contributing to the increase in chlorophyll content. However, it is important to note that excessive salt stress can ultimately harm the plant, leading to stunted growth and reduced productivity.

Arslanbey genotype showed the best results for the contents of Ch a, Ch b and carotenoids (0.74, 0.30 and 0.89, respectively). Among the treatments, the NaCl + EbR + SA treatment group showed the highest content of Ch a, Ch b and carotenoids (0.61, 0.30 and 1.14, respectively).

Arslanbey and PI-304269 genotypes had the highest Ch a content (1.72 and 0.63, respectively) and Ch b content (0.72 and 0.25, respectively) in the control groups. PI-650142 genotype showed the highest Ch a content (0.66) in the NaCl + EbR treatment group and the highest Ch b content (0.42) in the NaCl + EbR + SA treatment group (Figs. 3a and 3b). This finding indicates that the hormonal treatment of plants under NaCl stress led to a decrease in stress levels. This implies that plants experiencing salt stress are less affected by the stress following hormonal treatment (SA and/or EbR + SA). Arslanbey and PI-650142 genotypes showed the highest carotenoid content (1.53 and 1.55, respectively) in the NaCl + EbR + SA treatment group. PI-304269 genotype could not stabilise and NaCL treatment indicated the highest carotenoid content after the control group (Fig. 3c). This increase in carotenoid content can be considered a response to salt stress.

Arslanbey and PI-650142 genotypes showed the highest total chlorophyll content (3.01 and 2.53) in NaCI + EbR + SA treatment group (Fig. 3d). PI-304269 genotype showed a lower chlorophyll content in all treatment groups as compared to the control group. NaCl + EbR + SA treatment led to 170% and 263% increase in chlorophyll content in plants of Arslanbey and PI-650142 genotypes. In contrast, for the same treatment group, PI-304269 genotype plants showed a 60% decrease in the total chlorophyll content. A previous study reported that the chlorophyll content of camelina under salt stress differed according to the variety. The total chlorophyll content started to decrease at 100 mM salinity in Cheyenne and Suneson cultivars and at 150 mM salinity in Blaine Creek cultivar (Morales et al. 2017).

Chlorophylls are the critical components of photosynthesis, the process by which plants produce energy from sunlight. Therefore, alterations in chlorophyll content can substantially affect plant growth and development. Carotenoids are crucial molecules that act as antioxidants and help neutralise harmful free radicals. Treatment of salt stress–exposed plants with SA can increase their carotenoid content, which subsequently enhances the plants’ ability to mitigate the negative effects of the stress (Azooz, 2009). This may occur because SA treatment helps limit the toxic effects of chloride and sodium ions and exerts a protective effect against oxidative stress induced by salinity (Razavizadeh 2015). SA treatment could increase chlorophyll content, which could also help protect plants from the harmful effects of salt stress. These findings suggest that SA treatment could be a useful strategy to improve plant resilience to environmental stress. It was unable to find literature specifically reporting the effect of EbR and/or EbR + SA on salinity stress in the camelina plant. It seems that this topic has not yet been studied extensively in camelina plants or the related studies are yet to be published. However, we found some studies that investigated the effect of EbR on salt stress in related plants such as mustard and rapeseed; the findings of these studies may provide some insights into the potential effects of EbR on camelina. For example, foliar application of SA (1 mM) and EbR (0.1 mM) on black mustard increased chlorophyll content, particularly under moderate and severe salinity conditions. Baghizadeh et al. (2014) reported that SA treatment (1 mM) increased the contents of photosynthetic pigments (Ch a, Ch b and carotenoids) as compared to that in rapeseed plants under salinity stress. Furthermore, EbR enhanced photosynthesis and increased chlorophyll content in stressed plants, while SA enhanced plant stress tolerance and improved plant growth (Ghassemi-Golezani et al. 2020). The combination of these two compounds may exert synergistic effects and enhance their individual effects, thereby leading to an increase in chlorophyll content in the plant and potentially improving its growth and stress tolerance under salt-stress conditions.

The application of EbR through the root growth medium is reported to be an effective method to mitigate the harmful effects of salt stress on the growth and development of various plant species, ultimately leading to improved crop yields. For instance, Kagale et al. (2007) found that the addition of EbR (1 and 2 mM) to the germination medium markedly reduced the negative effect of salt stress on the seedling growth of Brassica napus. Similarly, the addition of 1 mM EbR to the root growth medium significantly accelerated the growth of sorghum (Sorghum bicolor) seedlings under salinity stress (150 mM) (Vardhini and Rao 2003). These studies suggest that the supplementation of the plant growth medium with EbR can substantially alleviate the adverse effects of salinity on plants grown under salt stress by regulating their critical physiological processes. However, it is important to note that this conclusion is based on a limited number of studies, and optimising EbR concentrations and application periods might be necessary to achieve these effects. Asraf et al. (2008) highlighted other issues that should be considered while using EbRs as root supplements. A critical issue is that the addition of EbRs to the field soil may not be effective as they can be degraded partially or entirely by soil microorganisms. Moreover, the addition of EbRs to the soil at optimal concentrations may not be practical from an economic perspective because of their high cost.

For most agronomic crops, the cost of using EbRs as soil supplements outweighs their yield benefits. Hence, more research is needed to optimise the efficiency of using EbRs as soil supplements for mitigating the negative effects of abiotic stresses such as salinity.

The supplementation of the plant growth medium with SA is also an effective method to mitigate the harmful effects of salt stress in various plant species. For instance, in Arabidopsis, the addition of 0.5 mM SA to a salt medium of 100 mM led to improved seed germination (Rajjou et al. 2006). Similarly, in tomatoes, SA treatment resulted in a fourfold increase in the growth rate and significantly higher photosynthetic activity under saline conditions. SA treatment was also shown to reduce MP-related damage caused by salt stress (Stevens et al. 2006). Another study demonstrated that the addition of 1 mM SA to soil with salinity ranging from 0 to 150 mM increased the photosynthetic capacity of wheat (Arfan et al. 2007).

Thus, treatment of the soil with SA seems to have a beneficial effect on improving seed germination as well as early and late vegetative growth in diverse plant species cultivated in saline environments. Therefore, SA could be considered a safe and eco-friendly compound that could enhance crop protection and promote crop yield in the agricultural sector. However, a limited number of studies have investigated the advantageous effects of SA on crop production under field conditions.

Figure 3.

The present study showed that the application of SA to camelina plants exposed to salt stress increased RWC and carotenoid levels as compared to those in the control group. This increase in RWC was observed in all genotypes, while the increase in the carotenoid content was noted only in PI-650142 genotype. Furthermore, the treatment of plants under salt stress with EbR mitigated the negative effects of the stress by improving plant growth, water content and plant survival. EbR treatment increased PWC and the contents of Ch a, Ch b and carotenoids in PI-650142 genotype as compared to those in other genotypes. Moreover, all genotypes showed an increase in RWC as compared to the control group.

The combined treatment of SA and EbR can potentially have synergistic effects on the camelina plant exposed to salt stress. The synergistic effects of these compounds could lead to even higher water content in the plant as well as improved growth and survival under salt-stress conditions. However, further studies are required to fully understand the mechanisms and effects of these compounds when applied together. The present study showed that the carotenoid content of the stress-induced plants treated with the EbR + SA combination increased as compared to that of the control group. Moreover, PI-650142 genotype showed an increase in DW and Ch a and Ch b contents. The RWC was increased in PI-304269 and PI-650142 genotypes, while the carotenoid content was increased in Arslanbey and PI-650142 genotypes.

In conclusion, SA and EbR have different but complementary effects on the camelina plant exposed to salt stress. When applied together, these compounds have the potential to provide even greater benefits in terms of PWC, growth and plant survival under these stress conditions. Further studies are recommended considering the sensitivity of genotypes with low salt tolerance.

Data availability

This manuscript has not been published and is not under consideration for publication elsewhere. All authors have approved of and have agreed to submit the manuscript to this journal.

Competing interests The authors reported no potential conflict of interest.

Acknowledgments

I am grateful to Ege University Planning and Monitoring Coordination of Organizational Development and Directorate of Library and Documentation for their support in editing and proofreading service of this study.

Funding No

Compliance with ethical standards

Ethical approval Not applicable

Consent to participate Not applicable

Consent to publish Not applicable

Ahmad P, Nabi G, Ashraf M (2011) Cadmium-induced oxidative damage in mustard [Brassica juncea (L.) Czern. & Coss.] plants can be alleviated by salicylic acid. South Afr J Bot 77(1):36–44. https://doi.org/10.1016/j.sajb.2010.05.003
Agarwal S, Sairam RK, Srivastava GC, Meena RC (2005) Changes in antioxidant enzymes activity and oxidative stress by abscisic acid and salicylic acid in wheat genotypes. Biol Plant 49(4):541–550. https://doi.org/10.1007/s10535-005-0048-z
Ali B, Hayat S, Fariduddin Q, Ahmad A (2008) 24-epibrassinolide protects against the stress generated by salinity and nickel in Brassica juncea. Chemosphere 72(9):1387–1392. https://doi.org/10.1016/j.chemosphere.2008.04.012
Arfan M, Athar HR, Ashraf M (2007) Does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in two differently adapted spring wheat cultivars under salt stress? J Plant Physiol 164(6):685–694. https://doi.org/10.1016/j.jplph.2006.05.010
Ashraf M, Athar HR, Harris PJC, Kwon TR (2008) Some prospective strategies for improving crop salt tolerance. Adv Agron 97:45–110. https://doi.org/10.1016/S0065-2113(07)00002-8
Azooz MM (2009) Foliar application with riboflavin (vitamin B2) enhancing the resistance of Hibiscus sabdariffa L. (Deep Red Sepals Variety) to Salinity Stress. J Biol Sci 9(2):109–118. https://doi.org/10.3923/jbs.2009.109.118
Baghizadeh A, Salarizadeh MR, Fjijas A (2014) Effects of salicylic acid on some physiological and biochemical parameters of Brassica napus L. (Canola) under salt stress. Int J Agricultural Sci 4(2):147–152
Bajguz A (2010) An enhancing effect of exogenous brassinolide on the growth and antioxidant activity in Chlorella vulgaris cultures under heavy metals stress. Environ Exp Bot 68(2):175–179. https://doi.org/10.1016/j.envexpbot.2009.11.003
Bajguz A, Hayat S (2009) Effects of brassinosteroids on the plant responses to environmental stresses. Plant Physiol Biochem 47(1):1–8. https://doi.org/10.1016/j.plaphy.2008.10.002
Ben Ahmed H, Abidi F, Manaa A, Mimouni H, Ezeddine ZID (2009) Salicylic acid induced changes on some physiological parameters in tomato grown under salinity. UC Davis, Department of Plant Sciences. Web. https://escholarship.org/uc/item/5j07c5vj. Accessed 26 March 2023
De la Reguera E, Veatch J, Gedan K, Tully KL (2020) The effects of saltwater intrusion on germination success of standard and alternative crops. Environ Exp Bot 180:104254. https://doi.org/10.1016/j.envexpbot.2020.104254
El-Khallal SM, Hathout TA, Ashour AA, Kerrit AA (2009) Brassinolide and salicylic acid induced growth, biochemical activities and productivity of maize plants grown under salt stress. Res J Agric Biol Sci 5(4):380–390
Ghassemi-Golezani K, Hassanzadeh N, Shakiba MR, Esmaeilpour B (2020) Exogenous salicylic acid and 24-epi-brassinolide improve antioxidant capacity and secondary metabolites of Brassica nigra. Biocatal Agric Biotechnol 26:101636. https://doi.org/10.1016/j.bcab.2020.101636
González L, González-Vilar M (2001) Determination of relative water content. In: Reigosa Roger MJ (ed) Handbook of plant ecophysiology techniques. Springer, Berlin, pp 207–212
Göre M, Göre M (2023) Salt sensitivity and some physiological and morphological adaptation mechanisms to salt stress in camelina. J Elementology. https://doi.org/10.5601/jelem.2022.27.4.2346. (1/2023).
Gugel RK, Falk KC (2006) Agronomic and seed quality evaluation of Camelina sativa in western Canada. Can J Plant Sci 86(4):1047–1058. https://doi.org/10.4141/P04-081
Gunes A, Inal A, Alpaslan M, Eraslan F, Bagci EG, Cicek N (2007) Salicylic acid induced changes on some physiological parameters symptomatic for oxidative stress and mineral nutrition in maize (Zea mays L.) grown under salinity. J Plant Physiol 164(6):728–736. https://doi.org/10.1016/j.jplph.2005.12.009
Hamada K (1986) Brassinolide in crop cultivation. In: MacGregor P (ed) Plant growth regulators in agriculture. No-yaku Ko-gyo-kai, Japan, pp 190–196
Hayat S, Ali B, Hasan SA, Ahmad A (2007) Effect of 28-homobrassinolide on salinity-induced changes in Brassica juncea. Turkish J Biology 31:141–146
Houimli SIM, Denden M, Mouhandes BD (2010) Effects of 24-epibrassinolide on growth, chlorophyll, electrolyte leakage and proline by pepper plants under NaCl-stress. Eurasian J Biosci 4:96–104
Kagale S, Divi UK, Krochko JE, Keller WA, Krishna P (2007) Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses. Planta 225(2):353–364. https://doi.org/10.1007/s00425-006-0361-6
Karimi G, Ghorbanli M, Heidari H, Khavari Nejad RA, Assareh MH (2005) The effects of NaCl on growth, water relations, osmolytes and ion content in Kochia prostrata. Biol Plant 49(2):301–304. https://doi.org/10.1007/s10535-005-1304-y
Karlidag H, Yildirim E, Turan M (2011) Role of 24-epibrassinolide in mitigating the adverse effects of salt stress on stomatal conductance, membrane permeability, and leaf water content, ıonic composition in salt stressed strawberry (Fragaria×ananassa). Sci Hort 130(1):133–140. https://doi.org/10.1016/j.scienta.2011.06.025
Kaydan D, Yagmur M, Okut N (2007) Effects of salicylic acid on the growth and some physiological characters in salt stressed wheat (Triticum aestivum L). Tarim Bilimleri Dergisi 13(2):114–119. https://doi.org/10.1501/Tarimbil_0000000444
Khalid H, Kumari M, Grover A, Nasim M (2015) Salinity stress tolerance of Camelina investigated in vitro. Scientia Agriculturae Bohemica 46(4):137–144. https://doi.org/10.1515/sab-2015-0028
Khan W, Prithiviraj B, Smith DL (2003) Photosynthetic responses of corn and soybean to foliar application of salicylates. J Plant Physiol 160(5):485–492. https://doi.org/10.1078/0176-1617-00865
Kočová M, Rothová O, Holá D, Kvasnica M, Kohout L (2010) The effects of brassinosteroids on photosynthetic parameters in leaves of two field-grown maize inbred lines and their F1 hybrid. Biol Plant 54(4):785–788. https://doi.org/10.1007/s10535-010-0143-7
Kohli SK, Handa N, Sharma A, Gautam V, Arora S, Bhardwaj R, Alyemeni MN, Wijaya L, Ahmad P (2018) Combined effect of 24-epibrassinolide and salicylic acid mitigates lead (Pb) toxicity by modulating various metabolites in Brassica juncea L. seedlings. Protoplasma 255(1):11–24. https://doi.org/10.1007/s00709-017-1124-x
Liu J, Gao H, Wang X, Zheng Q, Wang C, Wang X, Wang Q (2014) Effects of 24-epibrassinolide on plant growth, osmotic regulation and ion homeostasis of salt‐stressed canola. Plant Biol 16(2):440–450. https://doi.org/10.1111/plb.12052
Lutts S, Kinet JM, Bouharmont J (1995) Changes in plant response to NaCl during development of rice (Oryza sativa L.) varieties differing in salinity resistance. J Exp Bot 46(12):1843–1852
Mandava NB (1988) Plant growth-promoting brassinosteroids. Annu Rev Plant Physiol Plant Mol Biol 39(1):23–52
Mohammadreza S, Amin B, Forogh A, Hossin M, Sorayya S (2012) Response of Brassica napus L. grains to the interactive effect of salinity and salicylic acid. J Stress Physiol Biochem 8(2):159–166
Morales D, Potlakayala S, Soliman M, Daramola J, Weeden H, Jones A, Kovak E, Lowry E, Patel P, Puthiyaparambil J, Goldman S, Rudrabhatla S (2017) Effect of biochemical and physiological response to salt stress in Camelina sativa. Commun Soil Sci Plant Anal 48(7):716–729. https://doi.org/10.1080/00103624.2016.1254237
Noreen S, Ashraf M (2008) Alleviation of adverse effects of salt stress on sunflower (Helianthus annuus L.) by exogenous application of salicylic acid: growth and photosynthesis. Pak J Bot 40(4):1657–1663
Rajjou L, Belghazi M, Huguet R, Robin C, Moreau A, Job C, Job D (2006) Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms. Plant Physiol 141(3):910–923. https://doi.org/10.1104/pp.106.082057
Razavizadeh R (2015) Protein pattern of canola (Brassica napus L.) changes in response to salt and salicylic acid in vitro. Biol Lett 52(1–2):19–36. https://doi.org/10.1515/biolet-2015-0012
Razeq FM, Kosma DK, Rowland O, Molina I (2014) Extracellular lipids of Camelina sativa: characterization of chloroform-extractable waxes from aerial and subterranean surfaces. Phytochemistry 106:188–196. https://doi.org/10.1016/j.phytochem.2014.06.018
Russo R, Reggiani R (2015) Seed protein in Camelina sativa (L.) Crantz var. Calena. Int J Plant Soil Sci 8(2):1–6. https://doi.org/10.9734/IJPSS/2015/19003
Shahbaz M, Ashraf M, Athar HUR (2008) Does exogenous application of 24-epibrassinolide ameliorate salt induced growth inhibition in wheat (Triticum aestivum L)? Plant Growth Regul 55(1):51–64. https://doi.org/10.1007/s10725-008-9262-y
Shahid MA, Pervez MA, Balal RM, Mattson NS, Rashid A, Ahmad R, Ayyub CM, Abbas T (2011) Brassinosteroid (24-epibrassinolide) enhances growth and alleviates the deleterious effects induced by salt stress in pea (Pisum sativum L). Aust J Crop Sci 5:500–510
Song S, Liu W, Guo S, Shang Q, Zhang Z (2006) Salt resistance and its mechanism of cucumber under effects of exogenous chemical activators. Ying Yong Sheng Tai Xue Bao 17(10):1871–1876
Stevens J, Senaratna T, Sivasithamparam K (2006) Salicylic acid induces salinity tolerance in tomato (Lycopersicon esculentum cv. Roma): associated changes in gas exchange, water relations and membrane stabilisation. Plant Growth Regul 49:77–83. https://doi.org/10.1007/s10725-006-0019-1
Sudhir P, Murthy SDS (2004) Effects of salt stress on basic processes of photosynthesis. Photosynthetica 42(4):481–486. https://doi.org/10.1007/S11099-005-0001-6
Tester M, Langridge P (2010) Breeding technologies to increase crop production in a changing world. Science 327(5967):818–822. https://doi.org/10.1126/science.1183700
Turner NC (1981) Techniques and experimental approaches for the measurement of plant water status. Plant Soil 58(1–3):339–366
Vidya Vardhini B, Seeta Ram Rao S (2003) Amelioration of osmotic stress by brassinosteroids on seed germination and seedling growth of three varieties of sorghum. Plant Growth Regul 41(1):25–31. https://doi.org/10.1023/A:1027303518467
Vinocur B, Altman A (2005) Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Curr Opin Biotechnol 16(2):123–132. http://doi.org/10.1016/j.copbio.2005.02.001
Visser EJ, Zhang Q, De Gruyter F, Martens S, Huber H (2016) Shade affects responses to drought and flooding – acclimation to multiple stresses in bittersweet (Solanum dulcamara L). Plant Biol 18(Suppl 1):112–119. https://doi.org/10.1111/plb.12304
Wu G-Q, Jiao Q, Shui Q-Z (2015) Effect of salinity on seed germination, seedling growth, and inorganic and organic solutes accumulation in sunflower (Helianthus annuus L). Plant Soil and Environment 61(5):220–226. https://doi.org/10.17221/22/2015-PSE
Zaki HEM, Radwan KSA (2022) Response of potato (Solanum tuberosum L.) cultivars to drought stress under in vitro and field conditions. Chem Biol Technol Agric 9(1). https://doi.org/10.1186/s40538-021-00266-z
Zhao S, Zhang Q, Liu M, Zhou H, Ma C, Wang P (2021) Regulation of plant responses to salt stress. Int J Mol Sci 22(9):4609. https://doi.org/10.3390/ijms22094609

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Mitigation of Salt Stress in Camelina sativa by 24-epibrassinolide and/or Salicylic acid Treatments

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Abstract

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Introduction

Materials and methods

Plant material and Treatments

Membrane Permeability (MP)

Chlorophyll-a, Chlorophyll-b and Carotenoids

Statistical and data analysis

Results and discussion

FW and DW

PWC

RWC

MP

Chlorophyll-a, chlorophyll-b and carotenoid

Conclusion

Declarations

References

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