Mitigate salt stress in maize using Ecklonia maxima seaweed extracts

Seaweed extract (SE) biostimulant, derived from Ecklonia maxima is popular now. And Salinity stress poses a significant threat to crop productivity, making it crucial to explore strategies that alleviate its adverse effects. This study, using Afrikelp® derived from Ecklonia maxima as an example, aims to investigat the impact on maize (Zea mays L.) plants subjected to salinity stress.

Introduction

Salt stress, one of the most prevalent abiotic factors, affects approximately 20 million hectares of arable land globally. This detrimental situation is further compounded by poor agronomic practices, such as over-fertilisation, deforestation, and the excessive use of agricultural water, leading to a yearly increase in salt-affected farmland. In 2013, approximately 25 % of the world’s irrigated land was salinized with a further increase of 10 % in 2020. Such rapid conversion of fertile soil into saline soil poses a significant threat to the sustainability of agricultural production, vegetative coverage, and forest restoration. Therefore, it is crucial to develop salt-stress mitigation strategies and gain a comprehensive understanding of the impact of salt stress on plants, including plant salt tolerance mechanisms.

Seaweed extracts (SE) are among the promising eco-friendly strategies for mitigating salinity-related issues in crop production. They serve as a viable substitute for chemical fertilisers and belong to the category of plant biostimulants, commonly employed to improve crop yield and quality. They contain several phytochemicals, like amino acids, phytohormones, phenolics, polysaccharides and phycocolloids including., fucoidan, alginate, and carrageenan that can be beneficial to plants. In maize, SEs have been shown to alleviate salinity stress and improve growth and productivity. Considering that Zea mays (maize) accounts for approximately 12.4 % of the world’s food demand and ranks first in production volume worldwide (> 1.1 million tons/year), sustainable and eco-friendly strategies that improve its stress tolerance is of great importance for both yield stability and food security.The performance of SEs in mitigating plant stress is dependent on various factors such as the source of the seaweed species, the manner of processing the extract and the severity of the stress. Previous studies have shown the effectiveness of different seaweeds in alleviating salt stress in different crops. Someone studied the effect of SEs from Padina gymnospora on alleviating salinity stress in Solanum lycopersicum (tomato), while others investigated the impact of priming on salinity stress in Vigna sinensis (black-eyed peas) and Zea mays (maize) seedlings using three Egyptian seaweeds (Ulva fasciata, Cystoseira compressa, and Laurencia obtusa). As informed by our research to date, there are no studies that have investigated the effectiveness of commercial SEs from Ecklonia maxima in alleviating salt stress in maize. In addition, the mechanisms by which maize plants respond to such SE extracts under conditions of salinity stress have not been fully understood. In this context, the current study aims to investigate the effectiveness of commercial E. maxima extract biostimulant in mitigating the effects of prolonged salinity stress on maize plants by examining the associated morphological, physiological, biochemical and metabolic responses.

Materials and Methods

Commercial seaweed extract

The seaweed extract (SE) utilised was the commercially available SE Afrikelp® derived from the brown marine macroalgae Ecklonia maxima and prepared using a cold micronisation process.

Plant growth conditions and stress treatments

The mildly drought-resistant yellow maize cultivar that is commercially grown in the Western Cape was grown in a controlled growth room for 47 days (26 ℃ 30 % humidity). Maize seeds were planted in plastic 2 L pots filled with 500 mL of peat moss soil, with five seeds per pot. There were six treatments with four biological replicates per treatment and three plants per pot. The seeds were watered every 2 days with 100 mL (per pot) of half-strength Hoagland’s No 2 basal salt Mixture. Upon full expansion of the first leaf (10 days after sowing (DAS)), (V1), the half-strength Hoagland solution (already containing salt) was supplemented with additional saline for use in the relevant saline experimental treatments (see below: T3, T4, T5 and T6). At 14 DAS and upon the full expansion of the second leaf (V2), the experimental treatments received the relevant Afrikelp foliar applications (see below: T2, T4 and T6) (Fig. 1). The various experimental treatments were initiated as follows:

• T1 (Negative control): The plants were irrigated every second day with a half-strength Hoagland solution containing no supplementary salt. Additionally, plants were foliar sprayed with water at a rate of 2L/ha (12 mL/pot).

• T2 (Positive control): The plants received Afrikelp® foliar spray at a rate of 2 L/ha (12 mL/pot) in addition to being irrigated every second day with a half-strength Hoagland’s solution containing no additional salt.

• T3: The plants were irrigated every second day with a half-strength Hoagland’s solution supplemented with 200 mM NaCl. To prevent osmotic shock, the concentration of NaCl was increased by 50 mM per irrigation until 200 mM was reached.

• T4: The plants received SE Afrikelp® foliar spray at a rate of 2 L/ha (12 mL/pot) in addition to being irrigated every second day with a half-strength Hoagland’s solution supplemented with 200 mM NaCl.

• T5: The plants were irrigated every second day with a half-strength Hoagland’s solution supplemented with 300 mM NaCl. To prevent osmotic shock, the concentration of NaCl was increased by 50 mM per irrigation until 300 mM was reached.

• T6: The plants received SE Afrikelp® foliar spray at a rate of 2 L/ha (12 mL/pot) in addition to being irrigated every second day with a half-strength Hoagland’s solution supplemented with 300 mM NaCl.

Results

Impact of salt stress on the gas exchange parameters of SE treated and untreated maize plants

The gas exchange parameters of maize plants were analysed under salt stress and control conditions, both with and without the presence of SE. The results of the analysis are presented in Table 1, under the corresponding conditions, the photosynthetic rates (Pn) of plants treated only with SE (T2) and SE together with 200 mM salt stress conditions (T4) were 1.4-fold and 1.1 -fold higher, respectively, than that of untreated control plants (T1). The internal CO₂ concentration (Ci) values were significantly lower (p < 0.05) for SE treated plants under control conditions (T2) compared to untreated plants (T1). However, the opposite trend was observed in plants under salt stress conditions, where SE treated plants at 200 mM NaCl (T4) showed a significantly higher internal CO2 concentration (3.1-fold higher, p < 0.05) compared to plants treated only with 200 mM salt (T3). A similar pattern was observed for transpiration (E), where SE treated plants under control conditions (T2) had lower E rates (1.6-fold) than those of untreated plants (T1). However, under 200 mM salt stress conditions, the SE treated plants (T4) showed significantly (p < 0.05) greater transpiration rate compared to the salinity stress only treated plants (T3). For the stomatal conductance (gs), maize treated with water under control conditions (T1) showed significantly higher values compared to those treated only with SE (T2). In contrast, maize treated only with 200 mM salt (T3) showed significantly lower gs values than those that received SE with 200 mM salt treatment (T4). Compared to the aforementioned gas analysis parameters, there were no significant differences in leaf temperature (Tleaf) between the different treatments. There were differences in leaf vapour pressure deficit (VPDL) between the untreated plants (T1), SE treated plants (T2) and 200 mM salt only treatment (T3). However, there were no differences between 200 mM salt only treatment (T3) and salt stress together with SE treatment (T4). Gas exchange data for T5 and T6 plants treated were not obtained due to the severity of the 300 mM salt stress. The results also showed that under control conditions, both ChlA and ChlB concentrations were higher in SE treated plants (T2) compared to the other treatments (T1, T3, T4, T5 and T6).

Treatments	Pn (µmol/m2/s)	Ci (ppm)	E (mol/m2/s)	gs (mol/m2/s)	Tleaf ( °C)	VPDL (kPa).	ChlA (µg/mg FW)	ChlB (µg/mg FW)
T1	5.71±0.3	280.39±10.2	1.01±0.05	0.085±0.004	24.30±0.1	1.19±0.03	6.63±0.4	9.57±0.6
T2	8.28±0.5	145.92±8.4	0.60±0.03	0.049±0.003	24.38±0.2	1.24±0.04	8.46±0.5	11.27±0.7
T3	3.35±0.2	28.22±2.1	0.21±0.01	0.016±0.001	24.31±0.1	1.28±0.05	5.55±0.3	5.90±0.3
T4	6.53±0.4	89.96±5.6	0.45±0.02	0.035±0.002	24.29±0.1	1.27±0.04	7.70±0.4	9.01±0.5
T5	ND	ND	ND	ND	ND	ND	4.13±0.2	3.57±0.2
T6	ND	ND	ND	ND	ND	ND	4.71±0.3	5.67±0.3
P-value	0.0001	0.0000	0.0000	0.0000	0.6120	0.0000	0.0000	0.0000

Effect of salt stress on the morphological parameters and electrolytic leakage of SE treated and untreated maize plants

The analysis of morphological parameters, including total fresh biomass, fresh root weight, and dry root weight, demonstrated the significant growth-promoting effect of SE on maize plants under both non-stress (control) and salinity stress conditions (Fig. 2A-C). Maize plants subjected to intermediate (200 mM NaCl) and high (300 mM NaCl) salt concentrations without SE treatment (T3 and T5) displayed a markedly lower fresh biomass (28 % and 30 %, respectively), fresh root weight (33 % and 36 %, respectively), and dry root weight (36 % and 30 %, respectively) compared to those treated with SE (T4 and T6). These results were confirmed visually when examining the morphological changes to the maize leaves and shoots at the V2 stage under the different treatments (Figures S1 & S2). Furthermore, electrolyte leakage data indicated a significantly higher electrolyte leakage per gram dry weight (DW) in maize plants under salt stress conditions without SE treatment compared to those treated with SE under the same conditions (Fig. 3).

Hydrogen peroxide (H₂O₂) and MDA content in SE -treated and untreated maize plants under salt stress conditions

Under salt stress conditions, the H₂O₂ content in T3 (200 mM NaCl) and T5 (300 mM NaCl) plants (Table 2) increased at non-significant levels; 27 % and 30 % in respectively, compared to the control group (T1). However, the addition of SE to NaCl-stressed plants reduced this accumulation of H₂O₂, albeit at non-significant levels. Specifically, T4 plants (200 mM NaCl + SE) showed a 17 % decrease in H₂O₂ content, while T6 plants (300 mM NaCl + SE) showed a 12 % decrease compared to the salt stress controls (T3 and T5, respectively). Similarly, salt stress increased the lipid peroxidation product malondialdehyde (MDA) to a lesser extent in SE treated plants, relative to the untreated plants (Table 2).

Treatment	H2O2 (Mm/g DW)	Lipid peroxidation (µmol/g DW)	DPPH (TE mg/mg DW)	FRAP (TE mg/mg DW)
T1	0.07 ± 0.011	0.010 ± 0.0015	0.89 ± 0.134	0.39 ± 0.059
T2	0.05 ± 0.0075	0.010 ± 0.0015	0.93 ± 0.140	0.40 ± 0.060
T3	0.10 ± 0.015	0.012 ± 0.0018	0.74 ± 0.111	0.35 ± 0.053
T4	0.08 ± 0.012	0.009 ± 0.00135	0.77 ± 0.116	0.44 ± 0.066
T5	0.11 ± 0.0165	0.013 ± 0.00195	0.55 ± 0.083	0.25 ± 0.037
T6	0.09 ± 0.0135	0.013 ± 0.00195	0.80 ± 0.120	0.27 ± 0.041
P-value	0.0109	0.0020	0.0086	0.0087

Metabolic profiling of maize plants treated with SE and untreated plants with and without salt stress

To investigate the metabolomic response of maize to salt stress and SE treatment. In total, 86 metabolites were quantified from the shoot and root samples. An ANOVA of the shoots identified 47 responsive primary metabolites under different treatment conditions (T1-T6) and 34 responsive metabolites in the roots (FDR p-value < 0.05). Chemometric modelling methods such as principal component analysis (PCA) and hierarchical clustering analysis (HCA) heatmaps revealed treatment-related clustering in shoots data (Fig. 4). Interestingly, in the score space (Fig. 4A), the shoot samples treated with SE under 200 mM salt stress (T4) grouped more closely to the control samples (T1-T2); pointing to more similar metabolic profiles. The 300 mM salt stress (T5) clustered separately from SE and 300 mM salt stress (T6) which overlapped with the 200 mM salt stress only (T3) (Fig. 4). Moreover, the relative quantification via HCA heatmap showed a general increase of primary metabolites (namely sugars, amino acids, and organic acids) in the shoots under salt stress, specifically at the higher concentration (300 mM) (Fig. 4B).

To investigate the effect of SE Afrikelp® in shoots under control (no stress) condition, a volcano plot (p-value threshold = 0.06, fold change threshold = 2) was used (Fig. 5A). The statistically significant metabolites discriminating between the two groups (T1 vs T2) included ribose, sucrose, maltitol and glycerol (Fig. 5A). The levels of ribose, glycerol and maltitol decreased in SE treated shoots compared to untreated shoots, while the level of sucrose increased (Fig. 5B-E).

Under salt stress conditions, the relative quantification showed that salt stress elevated the levels of primary metabolites (amino acids, sugars, and organic acids) in 200 mM salt stress only (T3) shoots. However, in SE and 200Mm (T4) treated shoots, the levels of these primary metabolites were decreased (Fig. 6A-B). The elevated metabolites in 200 mM salt stressed shoots (T3) included sucrose, trehalose, raffinose, proline, alanine, tyrosine and phenylalanine. Contrastingly, these same metabolites were reduced in 200 mM salt and SE -treated shoots (T4) (Fig. 6). Moreover, the overall metabolite profile of 200 mM salt stress and SE – shoots (T4) was more similar to the untreated control shoots (T1) compared to the 200 mM salt stressed shoots only (T3) (Fig. 6).

The heatmap diagrams generated from the roots-only samples (Fig. 7B) showed slightly contrasting patterns to shoots. The PCA and the HCA of the roots data showed that the metabolic profile of salt stressed roots treated with SE is more similar to the metabolic profile of salt stressed roots with no SE treatment. A general decrease of primary metabolites was observed under salt stress conditions (with and without SE ) (Fig. 7A-B). However, key noticeable amino acids alterations were observed as evidenced by the levels of proline, alanine, pyroglutamic acid and glutamic acid which were increased in the salt stress untreated roots samples and decreased in SE -treated roots (Fig. 7C). Sugars such as sucrose, glucose, raffinose and cellobiose showed a decrease in roots in response to salt stress whereas ribose, maltose, lyxose and xylitose showed an increase in roots under salt stress (Fig. 7D).

To further understand the correlation between the measured primary metabolites and the morphophysiological measurements, correlation analysis was performed using shoots datasets (Fig. 8). The correlation analysis revealed that the growth promotion indicators of shoots were strongly and negatively correlated with salt stress only treatments where the accumulation of amino acids under salt stress is associated with a decrease in total biomass, root biomass and chlorophyll content (Fig. 8A). The correlation analysis of salt stressed shoots vs salt stressed with SE -treated shoots, showed a similar negative correlation, however with a deceased magnitude in the accumulation of amino acids and sugars in the latter. Contrastingly, the stress indicators, H₂O₂ and lipid peroxidation showed a positive correlation to the amino acid content, meaning that the accumulation of amino acids under salt stress is positively associated with increase of H₂O₂ and lipid peroxidation (Fig. 8B).

Hormone content in SE -treated and untreated maize plants under salt stress conditions

When we compare the level 3-indole acetic acid (IAA) in shoots (Fig. 9A), a significant increase in the SE treatment only (T2) was observed when compared to the 200 mM and 300 mM salt stress only treatments (T3 and T5). In the roots (Fig. 9B), IAA was significantly increased in the SE treatment (T2) compared to the 200 mM and 300 mM salt stress treatments only (T3 and T5). However, there was no difference in the SE and 200 mM salt treatment (T4) when compared to the 200 mM salt stress treatment (T3).

In the shoots (Fig. 9A), there was no significant difference in phenylacetic acid (PAA) between the control (T1) and SE Afrikelp® (T2), 200 mM salt treatment (T3) and the SE and 200 mM salt treatment (T4). However, PAA was significantly reduced in the 300 mM salt only treatment (T5) relative to T1, T2, and T4. In the roots (Fig. 9B), a significant decrease in PAA for the 200 mM and 300 mM salt only treatments (T3 and T5) was observed when compared to the control (T1) and the SE only treatment (T2). There was no difference in PAA in the SE with 200 M salt treatment (T4) when compared to the respective salt treatment only (T3).In the shoots (Fig. 9A), abscisic acid (ABA), except for 300 mM salt stress only treatment (T5), there was no significant difference between the treatments (T2, T3 T4 and T6) compared to the control (T1). An increase in ABA was observed upon salt stress treatment with or without SE with a similar trend and observation seen for ABA in the roots (Fig. 9B).

Discussion

Salt stress poses a significant threat to agriculture due to its detrimental impact on crop development and quality, making it one of the most challenging abiotic stresses. Consequently, it is crucial to adopt practices and innovations that can mitigate the negative consequences of salt stress to ensure satisfactory crop yields. It is suggested that the abundance of various bioactive compounds such as auxins, kinetin, cytokinin, zeatin, gibberellins and polyphenols in seaweed species help mitigate against various abiotic stress in addition to improving plant growth.

The findings of this study revealed a significant impact of SE treatment on the photosynthetic (Pn) rate of maize plants treated with SE (T2). Under salt stress conditions and treatment (T4), the maize plants demonstrated comparable levels of photosynthetic activity to the controls (T1 and T2), highlighting the beneficial effects of SE in mitigating the adverse effects of salt stress. This was also validated by the chlA and chlB contents which were significantly higher in SE only treatment (T2) and had the same trend as observed for the Pn rates. These results align with previous observation in the literature demonstrating that the application of SE can enhance the photosynthetic potential of plants by improving chlorophyll concentration in shoots. They observed an increase in cellular metabolic rate and higher values of electron transport rate (ETRmax) compared to untreated plants suggesting a potential enhancement in the translocation rate of molecules produced through photosynthesis to the fruit, leading to increased fruit yields.

Salt stress was also shown to negatively affect agronomic or morphological parameters of maize plants including height, root length, fresh biomass, fresh root weight, and dry root weight The inhibitory effects of salt stress on maize growth are due to its impairment of factors involved in plant metabolism, including osmotic adjustment, nutrient uptake, as well as protein and nucleic acid synthesis. It was noticeable that SE treatment could minimise the negative effects of salt stress at 200 mM and slightly minimize these effects at 300 mM salt (Fig. 2). This indicates that SE application can dampen the negative effects of high salt stress.

Under normal conditions, SE biostimulant showed a decrease in the levels of statistically significant metabolites namely ribose, glycerol and maltitol and showed an increase in the levels of sucrose. Ribose is known to be involved in plant processes such as photosynthesis, cell division, RNA formation and energy metabolism. However, the relationship between ribose levels in shoots and plant growth promotion is not clearly understood. Glycerol is involved in plant stress response where it has been shown that exogenous application of glycerol inhibits primary root growth and altered lateral root development in Arabidopsis thaliana. When plants need energy, stored lipids can be broken down into glycerol and fatty acids, resulting in accumulation of glycerol. In this study, low levels of glycerol were found to in SE -treated plants under normal conditions. This could mean that since the plants were not under stress, the breakdown of triglycerides was not required and instead, triglycerides formation might be encouraged. Sucrose levels on the other hand, were found increased in the SE -treated plants compared to the control. Sucrose has been identified as a dominant regulator of growth processes in plant development. As such, the observed accumulation of sucrose in SE -treated plants can be positively associated with plant growth promotion mediated by SE biostimulant.

We also investigated the effect of the SE and salt stress treatments on plant hormone concentrations of IAA, PAA and ABA given their importance in plant growth and stress adaptation. The auxins, IAA and PAA, have a variety of roles in plants, specifically in stimulating or inhibiting root growth depending on the plant species and concentration. Upon salinity stress, no significant differences for both IAA and PAA were observed in the maize shoots but were significantly decreased in the roots. However, there was no significant difference between treatments only with salt and the SE with salt stress treatments. ABA is a key hormone involved in plant adaptation to abiotic stresses. It regulates stomatal closure, reducing water loss and contributing to various developmental processes. In the shoots, a small but non-significant increase in ABA concentration was observed in the treatment with SE only and larger but non-significant ABA increases with the salt stress treatments (including SE). The ABA increase in the leaves could explain in part the observed lower stomatal conductance and transpiration rates for the same treatments. ABA increase would therefore promote stomatal closure to reduce water loss in response to salt stress and is supported by previous studies.

Conclusion

In conclusion, Ecklonia maxima seaweed extracts treatment alleviates some of the adverse effects of salt stress on maize plants. Ecklonia maxima seaweed extracts treatment significantly improved photosynthetic capacity and preserved cell membrane integrity to maintain plant functioning under salinity stress. Additionally, Ecklonia maxima seaweed extracts treatment resulted in metabolic reprogramming of primary metabolites such as amino acids, sugars and organic acids associated with stress alleviation and adaptation to salt-induced stress. Typically, biostimulant treatment in agricultural applications occurs as pre-treatment in anticipation of future abiotic stress events, we show that application of Ecklonia maxima-derived biostimulant of maize subject to salt stress still mitigates the associated negative effects on plant growth. Lastly, the potential of Ecklonia maxima seaweed extracts biostimulant treatment to mitigate salt stress in other important commercial crops should be investigated, especially in the context and differences between C2 and C4 plants as a solution for salt-compromised agricultural soils.

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