Brassinolide and Zinc Effect on Physio-Biochemistry of Garden Pea (Pisum sativum L.) under Water Deficit Condition

An Indian consume 15.4 kg of pulses annually. Pulses contain protein, fibre, minerals, nutrients and physiologically active compounds (Behera et al., 2020). The UN and FAO named 2016 the “International Year of Pulses,” calling pulse seeds “nutritious seeds for a sustainable future” to boost pulse demand and supply. Biotic and abiotic stresses considerably affect pulse crop yields worldwide (Pavithra et al., 2020). Water shortages affect pulse production most (Toker and Mutlu, 2011). Arhar, chickpea, gram and other pulses consumption are getting increased among the agricultural foods. Garden pea being an important pulse crop gained utmost attention by growers and consumers due to their quick growth habit, requiring low input costs, great yields, nitrogen fixation and versatility in fitting in any cropping system. This makes garden pea a formidable crop for agricultural diversification. Pea seeds are an excellent source to obtain daily intake of protein (23-25%), fibre (5%), soluble carbohydrates (5%), vitamins (B, K and E) and minerals (many of which are not found in other foods) (Cervenski et al., 2017; Zilani et al., 2017). Peas are laxative, anti-diabetic, antioxidant, antifungal, antibacterial and anticancer in nature (Mejri et al., 2019). Irrigation water is scarce worldwide in agriculture (Cai et al., 2011). Unreliable precipitation has caused drought in India. Closing stomata reduces transpiration, helping plants during drought (Copolovici et al., 2014; Nabi et al., 2019). Insufficient soil moisture, especially during flowering and pod-filling affects pea crop yields and may influence protein-to-starch ratio (Cervenski et al., 2017).
       
Brassinosteroids (BRs) regulate male sterility and other physiological processes (Cervenski et al., 2017). Since rape plant pollen first identified BRs, various research has investigated their role in plant physiology i.e., flower buds, pollen, fruit, seeds and leaves had phytohormone (Hussain et al., 2020).  These substances can also be free molecules, glucosides, or sulphates which are necessary for plant growth. Phytohormones are crucial in drought settings (Hussain et al., 2020). This bio-regulator also interacts with other phytohormones to activate antioxidant enzymes and produce osmo-protectants (Zou et al., 2018).
       
There are 28-homobrassinolide and 24-epibrassinolide (e-BL) are commercially available BR isomers with similar actions. Plants need mineral nutrients for metabolism and signal transduction particularly zinc (Zn). It promotes aldolase and carbonic anhydrase. Zn is needed to make lipids, proteins, auxin co-factors and nucleic acids in plants. Zn treatment improves crop yield and quality (Chattha et al., 2017; Hassan et al., 2019), but Zn deficiency lowers yield and quality (Chattha et al., 2017; Hassan et al., 2019). Zinc toxicity prevents cell division and elongation, reducing biomass production. According to research, zinc in small doses may benefit wheat, sunflowers, tomatoes and red cabbage (Sadoogh et al., 2014, Eslami et al., 2014). As per studies, e-BL and zinc reduce drought negative effects. However, this raises questions about their modes of action and how they affect plant physiological and biochemical processes to make them drought-resistant. In light of this hypothesis, this article examines the effects of drought stress on plants and draws attention to the knowledge gaps in plant physiology, biochemistry and molecular biology regarding the effects of water deficit stress at different growth stages in garden pea and the effects of e-BL, Zn and their combination.

The experiment was conducted in the poly house of Department of Horticulture at Institute of Agricultural Sciences, Banaras Hindu University during rabi seasons of 2016-17 and 2017-18. The growing season comprised of average temperature of 20°C and 40% relative humidity. The experimental setup under the poly house shielded it from the rain. The Department of Genetics and Plant Breeding supplied the garden pea var. HUP-2 seeds and the Department of Plant Physiology of the institute supplied e-BL and other biochemical analysis chemicals for the experiment. Uniform-sized seeds were treated in three surface-sterilized petri plates. One petri plate contained water, whereas the other two contained e-BL at 0.05mM and 0.01mM (Pradhan et al., 2018). Pre-soaked seeds were planted in pots with 3 kg of soil and fertilizer and zinc. Only three seedlings from each container were transferred.
       
The seedlings were nurtured at the ideal moisture level. Plants were irrigated every alternate day. Stopping irrigation marked the beginning of water shortage therapy after 40 days assessment beginning with planting seeds till these are fully grown. Irrigation with tap water was performed every alternate day on the plants that were under control. Both water deficit patterns and two dosages of e-BL and/or Zn were employed in the treatment. The treatment comprised of Water deficiency before anthesis (WDBA), water deficiency after anthesis (WDAA), WDBA + 0.01 mM e-BL, WDBA + 0.05 mM e-BL, WDBA + Zn, T₀ (control), T₁, T₂, T₃, T₄, T₅, T₆, T₇ and T₈.
       
Three completely randomized trials made up the experiment. The experiment repeated three times with each treatment and with calculated 5% significance of mean differences were calculated. Sairam (1994) method of membrane stability index (MSI) calculation was adopted. One sample with 0.2 mg of leaf was used in 10 ml of sterile water and tested for electrical conductivity (EC1) after 30 minutes at 40°C and EC2 was measured for other sample after 10 minutes at 100°C. The formula of MSI calculation as followed;
   
The relative water content (RWC) was estimated by using the protocol of Dionisio-Sese and Tobita (1998).
   
Where,
FW= Fresh mass,
TW= Turgid weight,
DW= Dry weight.
       
Proline was estimated by method described by Bates et al., (1973). A mortar and pestle homogenized 0.5 g of leaves in 5 ml of 3% sulphosalicylic acid for five minutes. The supernatant was preserved after 6000 g RPM for 10 minutes. 5 ml of a 3% sulphosalicylic acid aqueous solution extracted the residue three times. 15 ml of supernatant portions were mixed in a test tube contained 2 ml of extract, glacial acetic acid and ninhydrin reagent. After 30 minutes in a 100°C water bath, the reaction mixture turned brick red. After cooling, the reaction mixture was added 5 ml of toluene and passed to a separating funnel. The absorbance of mixture and toluene blank were compared by using spectrophotometer at 520 nm. Proline was measured proline in each plant sample using a standard curve.
       
The level of superoxide dismutase (SOD) activity was determined by following methods described by Dhindsa et al., 1982. 3 ml of the reaction mixture containing 0.1 ml of 1.5 M sodium carbonate, 0.2 ml of 200 mM methionine, 0.1 ml of 2.25 mM NBT, 0.1 ml of 3 mM EDTA, 1.5 ml of 100 mM potassium phosphate buffer, 1 ml of dimethyl sulfoxide and 0.1 ml of enzyme extract were placed in test tubes in duplicate. In order to serve as a control, two tubes were used, that were devoid of enzyme extract. After inserting the tubes for 15 minutes in the presence of two fluorescent lights rated at 15 W, the reaction was kicked off by adding 0.1 ml of riboflavin at a concentration of 60 M to each of the tubes. It was possible to put a stop to the reaction by turning off the light and covering the tubes with a dark cloth. The colour was obtained by the tubes that did not contain any enzyme extract. A non-irradiated, complete combination that did not create any colour was utilized as a blank for the experiment. The absorbance was measured at 560 nm and one unit of enzyme was utilized as the amount of enzyme. This resulted in lowering of sample absorbance in comparison to the sample that did not contain enzyme. The EU was expressed as per g fresh weight basis, by using the following formula:  
(+) and (-) indicated with enzyme and without enzyme respectively. H₂O₂ content was measured in the first fully grown leaf of stressed and normal plants by Mukherjee-Choudhary method (1983). 10 mL of cold acetone was mixed with 0.1g of leaf sample. Whatman No. 1 filter paper filtered the homogenate. First 4 ml of titanium reagent and then 5 ml of concentrated ammonium solution were added to the extract to precipitate the peroxide-titanium complex. 10,000 rpm centrifugation lasted 5 minutes by dissolving the precipitate in 10 ml of 2% sulfuric acid after discarding the supernatant. After spinning to remove undissolved particles, absorbance was measured at 400 nm against a blank (2N H₂SO₄).

The common indicator that is often noticed under stressful situations was the leaf proline content presented in Fig 1. During the first year, the treatment T₈ showed a larger proline content when it was seen at 48 days after sowing (DAS). Later, during the second year, the treatment T₁₄ showed a larger proline content when it was noticed at 63 DAS. The results of the second year observations at 48 and 63 DAS revealed that the treatment T₅ contained significantly more proline than the other treatments. The lowest amount of proline found in either year samples with the control (T₀). Similar report also showing that the buildup of proline in response to drought stress may be caused by the promotion of its production, the inhibition of its breakdown, or the destruction of protein (Gomes et al., 2010). When compared with the control group, the administration of 4M-EBL resulted in a considerable increase in the proline content (Mohammadi et al., 2020). It has been observed that BRs increase the amount of proline in a substance by influencing the expression of genes involved in the process of proline biosynthesis (Talaat and Shawky, 2013). On the other hand, BRs encourage the production of nucleic acids via the process of biosynthesis in plant cells (Bajguz, 2000). Therefore, the rise in proline content that occurred after the application of EBL is most likely the result of an increase in the synthesis of nucleic acids in plant cells and their conversion into the necessary amino acids, such as proline. After the administration of EBL, it has been observed that the presence of BRs under stressful condition results in an increase in the amount of proline (Agami and Mohamed, 2013; Rady, 2011).
 

       
Garden pea was subjected to drought stress and the effect of 24-EBL and zinc on the membrane stability index (MSI) of the leaf as a percentage were recorded (Fig 2). The index of membrane stability in leaf tissue was shown to decrease as the amount of moisture stress increases. In the current experiment, this important sort of results in terms of membrane stability index was observed, which is consistent with the findings some researchers (Merwad et al., 2018). However, brassinosteroid was able to lessen the effect that water stress had on the permeability of the plasma membrane (Siddique and Husen, 2021; Sahoo et al., 2020). When compared with all the other treatments at 48 DAS, the post anthesis drought (T₅) exhibited the greatest impact. This was the case in the first year of the experiment. As the days progress, observations were made at 63 DAS and 85 DAS, it was observed that the effects of the treatment were decreasing while the control was practically increasing. In a similar manner, in the second year, LDS + 0.05 mM 24-EBL + Zn (T₁₅) had the optimal values in comparison to all the other treatments at the 48 DAS, but diminished effects were reported at later stage. The control had the greatest recorded relative water content (RWC) of leaf in the first 48 days after emergence (DAS), whereas the treatment T₂ had the highest RWC of leaf during 63 DAS and 85 DAS during the first year presented in Fig 3. Similarly, the RWC % was found to be more in the control plants during the first few days (48 DAS) and it rose at a rate that was comparable across treatments T₂ and T₇. Some study showing under conditions of extreme water stress, the seedlings that were treated with 0.2 mg/L 24-EBL had a leaf water content that was much higher than it was when they were properly watered (Li et al., 2012). The reason for this might be because there is a decrease in the amount of water that is lost from the leaf as a result of transpiration (Yavas and Unay, 2016). According to El-Khallal (2002), there is a possibility that brassinosteroids will regulate the expression of water stress-inducible proteins as well as induce the de-novo synthesis of specific polypeptides. This will encourage damaged plant cell membranes to recover and maintain homeostasis, which will result in higher RWC and a higher MSI.
       

 

 
Initially, it was observed that the activity of leaf superoxide dismutase (SOD) (Fig 4) was higher in control plants during the first 48 DAS. However, increased activity was found in treatment T₅ during the first 63 DAS and then the activity was once again higher in control during the first 85 DAS for the first year. However, results from the second year revealed that treatments T₅ and T₁₂ produced more activity at both 48 and 63 DAS. At 48, 63 and 85 DAS after treatment, various treatments such as T₃, T₈ and T₁₅ were shown to produce reduced activity. However, in the second year, treatments T₁₃ and T₁₀ had lower levels of activity at both 48 and 63 DAS. Reactive oxygen is produced and used by enzyme systems like catalase and SOD. Catalase and superoxide dismutase activities were stifled in pea plants that were put under the stress of drought (Li and Van Staden 1988 a, b). The use of BRs was shown in earlier studies to have a considerable impact on the antioxidant enzyme activity (Li and Van Staden, 1988 a, b). SOD activity was shown to be enhanced in rice after treatment with homo-brassinolide (Chen et al., 1997). Similar findings were seen in pea research conducted by (Alexieva et al., 2001). The results of this experiment also demonstrated the same kind of repercussions. The activities of CAT, POD, APX and SOD all increased in response to drought stress (Bajguz and Hayat, 2009), which suggests that drought stress circumstances (Sahoo et al., 2019) result in an inhibition of reactive oxygen species (Mohammadi et al., 2020). The lowest level of catalase activity was likewise seen in conditions of water stress (Sairam and Srivastava, 2001; Sahoo et al., 2021).
 

       
The amount of H₂O₂ found in leaf tissue was measured and documented in Fig 5 and Fig 6 for a number of different treatments under drought stress circumstances. In the beginning, the observations that were recorded demonstrated that treatment T₄, T₅, as well as T₁₃ and T₈, had relatively greater levels of H₂O₂ in the leaf at 48 DAS, followed by T₃, T₅, T₈ and T₁₃ on 63 DAS. The pre-anthesis drought treatment had the greatest H₂O₂ content at 48 DAS in the second year, but the greater quantity was found in treatment T₁₃ at 63 DAS, which is comparable to the original first observation made in the first year. During the first year, the treatment with 0.05 mM 24-EBL (T₂) produced the results with the lowest content. In addition, the treatment T₉ had the least amount of an impact at 48 DAS and later at 63 DAS, despite the fact that the control plants had the least amount of leaf H₂O₂. Catalase is an enzyme that is present in virtually all living creatures that are exposed to oxygen. It is responsible for catalysing the breakdown of H₂O₂ into water and oxygen. Catalase may be found in bacteria, plants and mammals. BR treatment resulted in a considerable drop in H₂O₂ concentration (Damghan, 2009). It would appear that the application of brassinosteroids during times of stress improved the plant’s ability to withstand drought by increasing the concentration of osmolites such as soluble sugars and proline. As a result, this aided in the maintenance of osmotic pressure within cells and led to an increase in the production of H₂O₂ (Zahediet_al2019).
 

 

Anti-oxidant activity in cell increases when it adopt protection mechanism under stress conditions which ultimately influence the yield-related parameters. The combination treatments with 24-EBL and zinc maintained osmolyte buildup and ROS reduction, enhancing garden pea production. This study suggests that 24-EBL may reduce the impact of drought stress in plant. This influences cell signaling, which may regulate several plant traits. Its expansion helps anti-oxidants, compatible solutes and photosynthetic pigments aggregate. 24-EBL alleviated moderately dry conditions in the pea genotype HUP-2. There is plenty of time to study this novel phytohormone and Its interaction with hormones and stress-related traits may help to generate drought-tolerant pea cultivars. However, Molecular and proteomic studies on mode of action of 24-EBL is crucial, as 24-EBL helps tissue-specific separation and plant component development.

This work was supported by fellowship from UGC. Laboratory facility was provided by Department of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi.

Jyostnarani Pradhan performed the experiment and wrote the first draft of the manuscript. Jyoti Prakash Sahoo, Laxmipreeya Behera, Kartik Pramanik, Siddhartha Shankar Sharma, and Jannila Praveena provided valuable information for preparation of the manuscript. Kailash Chandra Samal coordinated the process and gave valuable comments. All authors read and approved the final manuscript.

All data generated or analyzed during this study are included in this published article.

The Authors declare no conflict of interest.