Bangladesh Agron. J. 2023, 26(1): 75-83 ENHANCING SALT STRESS TOLERANCE AND YIELD PARAMETERS OF PROSO MILLET THROUGH EXOGENOUS PROLINE AND GLYCINE BETAINE SUPPLEMENTATION M.M. Alam and M. Hasanuzzaman* Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh. *Corresponding author, Email: mhzsauag@yahoo.com (Received: 13 July 2023, Accepted: 01 September 2023) Keywords: Proso millet, abiotic stresses, salinity, relative water content, ionic toxicity Abstract Proline (Pro) and glycine betaine (GB) act as significant osmoprotectants, potentially mitigating the detrimental effects of various abiotic stresses in plants. Given the growth-enhancing capabilities and other regulatory roles of Pro and GB, the current study was conducted to assess their function in imparting salt stress resilience in proso millet (Panicum miliaceum L). Proso millet plants were subjected to two levels of salt stress (S1 = 150 mM; S2 = 300 mM). Foliar applications of Pro (0.5 mM) and GB (0.5 mM) were provided under control and salt-stressed conditions at 10-day intervals twice. Compared to control conditions, plant growth, fresh and dry weight, leaf relative water content (RWC), SPAD value, and yield-contributing attributes noticeably decreased under salt stress. In contrast, plants supplemented with Pro and GB exhibited enhanced characteristics. Moreover, growth and yield parameters improved in salt- treated proso millet plants when supplemented with Pro and GB. These results suggest that the foliar application of Pro and GB can alleviate salt-induced oxidative stress in proso millet plants by modulating the antioxidative defense. Introduction Agricultural productivity across the globe is critically endangered by an array of abiotic stresses, such as drought, salinity, flooding, and extreme temperatures. Salinity, prevalent in over 100 countries, is particularly concerning as it degrades agricultural quality and yield, posing a significant environmental challenge (Dey et al., 2021; Sabagh et al., 2021). Studies reveal that nearly 20% of the Earth's arable land grapples with the harmful impacts of salt stress (Hasanuzzaman and Fujita, 2022). This stress leads to excessive absorption of sodium (Na+) and chloride (Cl−) ions, triggering ionic stress and cytotoxicity that severely hamper plant growth and development (Shah et al., 2021). Among the affected crops, proso millet (Panicum miliaceum L.), a drought-resistant variety, is significantly vulnerable to salinity stress. Despite its drought resistance, it demonstrates limited tolerance to elevated salt levels (Zou et al., 2019). High soil salinity upsets the plant's water balance, creating a water deficit and inhibiting nutrient uptake. The resulting osmotic stress from salt accumulation hinders growth, diminishes photosynthetic activity, and disrupts reproductive development (Akhter et al., 2021; Shah et al., 2021; Mushtaq et al., 2023). The escalation of oxidative damage due to reactive oxygen species (ROS) accumulation also results in cell membrane and cellular dysfunction (Hasanuzzaman et al., 2020; Saleem et al., 2021; Hasanuzzaman and Fujita, 2022). Managing salinity stress requires an integrated approach, incorporating agronomic practices, breeding initiatives, and innovative biotechnological tools. The development of salt- resistant varieties, refining irrigation techniques, and deploying effective soil management about:blank 76 Alam & Hasanuzzaman strategies are all steps towards mitigating the adverse effects of salinity stress. Additionally, researchers were exploring various biochemical strategies to amplify the salinity tolerance of proso millet, including the external application of compounds such as proline (Pro) and glycine betaine (GB). Pro, an osmolyte integral to osmotic adjustment and stress resistance in plants, and GB, a crucial chemical osmoregulator, have both shown potential in alleviating salinity stress. They contribute to maintaining cellular osmotic balance, protect cells from oxidative damage, regulate stress response enzymes, and enhance the overall salt tolerance of proso millet (Shafi et al., 2019; Hosseinifard et al., 2022; Zhu et al., 2022; Bai et al., 2022). However, the efficiency of Pro and GB is contingent on a myriad of factors such as concentration, method of application, and specific proso millet genotypes. With this in mind, this study was undertaken to evaluate the effects of applying Pro and GB under varying salinity stress levels in proso millet cultivation. Materials and Methods Plant materials and experimental design Healthy proso millet (Panicum miliaceum L.var. BARI Cheena-1) seeds were carefully chosen and consistently sown in 16 L plastic containers. Baseline applications of organic manure and chemical fertilizers, including urea, triple superphosphate, and muriate of potash, were used. Twenty days after sowing (DAS), along with the control group (0 mM NaCl), we introduced mild (150 mM NaCl) and severe salinity (300 mM NaCl) to the plants. Supplements of proline (Pro) (50 μM) and glycine betaine (GB) (50 mM) were given at 10 and 20 DAS under both normal and saline conditions. The study employed a completely randomized design (CRD) with three repetitions. Evaluation of plant height Proso millet plant heights were measured at 20, 30 DAS, and upon harvesting. A standard measuring scale was used to gauge the distance from the base level to the uppermost leaf tip of each plant. The average measurements were taken from five arbitrarily chosen plants from each pot. To calculate the Fresh Weight (FW) per plant, randomly uprooted three sample plants from each repetition and used a balance for weighing. After determining FW, the samples were dried in an oven at 80℃ for 48 h. Following this, they were weighed again to measure the Dry Weight (DW), with the average dry weight recorded as DW per plant. Assessment of SPAD values The SPAD meter (Minolta Camera Co., Osaka, Japan) was utilized to evaluate the top, middle, and base sections of five randomly selected leaves from each pot. Measurements from each section were then averaged (Yuan et al., 2016). Estimation of yield contributing parameters Standard procedures were employed to measure parameters contributing to yield, including the number of tillers per hill, panicle length, filled and unfilled grains per panicle, 1000- seed weight, and grain yield. Measurement of relative water content Five leaves were randomly chosen from each pot, cleaned, and weighed to determine the fresh weight (FW). The samples were then soaked in distilled water for 24 h, after which excess surface water was removed to measure the turgid weight (TW). The samples were then oven-dried Enhancing salt stress tolerance of proso millet by proline and glycinebetaine 77 for 72 h to obtain the dry weight (DW). The relative water content (RWC) was calculated using the formula: RWC (%) = (FW−DW)/(TW−DW) × 100 (Barrs and Weatherley, 1962). Statistical analysis The collected data from the three repetitions were statistically evaluated using Staistix 10 software. A one-way analysis of variance (ANOVA) was performed. Fisher's least significant difference (LSD) test was applied at the 5% significance level to compare the mean differences. Results and Discussion When subjected to S1 and S2, a reduction of 5% and 18% in plant height was observed at 30 DAS, respectively. This reduction progressively intensified over time, with the most significant decrease recorded during harvest—13% and 27% for S1 and S2, respectively. Nonetheless, the application of Pro and GB counteracted the adverse effects of salt stress on plant height. While the impact wasn't considerable at 30 DAS under both S1 and S2, these compounds proved more effective at 50 DAS. Pro, in particular, was highly successful in counterbalancing the detrimental effects of both levels of salt stress on proso millet. Table 1. Plant height at 30 DAS, 50 DAS, and at harvest of proso millet under salt stress supplemented by Pro and GB Treatment Plant height (cm) 30 DAS 50 DAS at harvest C 18.77±0.62 ab 50.72±0.97 a 74.60±1.39 a Pro 19.26±0.58 a 50.86±2.39 a 74.48±0.69 a GB 18.46±1.05 ab 51.36±0.99 a 75.22±0.72 a S1 14.71±0.52 ab 44.71±0.05 d 65.12±3.87 de S1+Pro 16.33±0.32 bc 49.22±2.34 b 71.02±1.52 bc S1+GB 15.63±0.86 bc 47.56±1.58 c 68.38±3.01 cd S2 11.82±0.24 e 39.08±0.58 e 54.55±4.91 f S2+Pro 14.25±0.51 cd 46.44±0.61 c 66.21±1.00 de S2+GB 13.26±0.74 d 44.93±2.19 d 63.28±4.59 e Plant morphological attributes, such as plant height, were significantly affected by salt stress. This is due to a decrease in cell growth, chlorophyll content, nutrient absorption, relative water content, transpiration rate, and stomatal conductance (Khalid et al., 2022; Rasool et al., 2023). However, the accumulation of Pro and GB can play a pivotal role in maintaining cellular osmotic balance, scavenging reactive oxygen species, and stabilizing proteins and membranes. Consequently, these osmo-protectants effectively counteract the adverse effects of salinity stress on plant growth, allowing plants to flourish even under demanding conditions (Desok et al., 2019; Dikilitas et al., 2020; Khalid et al., 2022). For instance, de Freitas et al. (2019) reported increased plant growth, gas exchange, and relative water content when Sorghum bicolor plants were sprayed with 30 mM of Pro under 75 mM of NaCl stress. Similarly, enhanced plant height and chlorophyll content were observed in Oryza sativa when treated with GB spray under salt stress (Annunziata et al., 2019). Moreover, Abbas et al. (2012) found that salt-stressed Citrus sinensis plants, sprayed with Pro (25 mg L−1), 78 Alam & Hasanuzzaman showed an increased production rate of salt-responsive proteins and a decreased salt impact on plant height and leaf numbers, aligning with our current findings. Under salt stress conditions, both fresh weight (FW) and dry weight (DW) decrease in a dose-dependent manner. When exposed to S1 and S2, FW reduced by 18% and 27%, respectively. However, the application of SA proved to be more effective under both S1 and S2, enhancing FW by 13% and 12%, respectively. A similar trend was observed for DW. The most significant decrease (34%) in DW occurred under S2, but SA and Pro were able to enhance it by 12% and 17%, respectively, under S1. Meanwhile, under S2, these agents could only increase DW by 17% and 12%, respectively. Fig. 1. Fresh weight (A) and dry weight (B) of proso millet under salt stress supplemented by salicylic acid and Pro.Here, Pro and GB represent Pro and GB, respectively. Elevated salinity levels can significantly negatively impact plants, leading to noticeable effects such as plant mortality and reduced productivity (Hannachi et al., 2022). Increased soil salinity can trigger osmotic stress, resulting in a prolonged water deficit within plant cells. At the same time, it can induce severe ion toxicity and oxidative damage, collectively inflicting detrimental effects on plant growth, which consequently reduces both the fresh weight (FW) and dry weight (DW) of the plants (Desok et al., 2019; Qaoud et al., 2023). As illustrated in (Figure 1), NaCl stress has noticeably reduced the assessed parameters in proso millet. This reduction could likely be due to two reasons. First, the presence of salt in the soil solution hinders water uptake from the plant root zone to the cell, leading to a decreased growth rate. Second, excessive salt infiltration through the transpiration streams can cause cellular injuries to the transpiring leaves. These injuries could potentially lead to further damage, resulting in diminished photosynthetic and growth capacities (Elhindi et al., 2017; Shehata and Nosir, 2019). However, Pro plays a pivotal role in maintaining ion homeostasis within plants. It helps mitigate the uptake of toxic ions such as sodium and facilitate the uptake of essential nutrients like potassium. This ion balance supports critical metabolic processes, bolsters energy production, and aids nutrient transport. As a result, it fosters increased biomass accumulation, leading to a higher FW and DW in plants (Ibrahim et al., 2019). Conversely, GB, a naturally occurring compound, has been found to significantly enhance plant growth and promote dry weight accumulation under salt stress conditions (Joushan et al., 2020). Enhancing salt stress tolerance of proso millet by proline and glycinebetaine 79 Fig. 2. SPAD value (A) and RWC (B) of proso millet under salt stress supplemented by salicylic acid and Pro. Here, Pro and GB represent Pro and GB, respectively. At 30 DAS, the SPAD value decreased by 11% and 17% under S1 and S2 conditions, respectively, compared to the control plants. However, the application of Pro and GB to stressed plants demonstrated a similar trend in mitigating salt stress-induced damage in proso millet. Proso millet was affected more severely by a higher salt stress dose, S2 (18%), than the lower dose, S1 (9%), in terms of RWC. However, the application of Pro and GB helped alleviate these negative effects. Pro was found to be more effective under lower levels of salt stress compared to the higher one. Under stress conditions, plants adopt an essential survival strategy by adjusting their photosynthetic capacity. This involves altering various photosynthetic attributes, including the SPAD value, to ensure their survival and optimal functioning despite adverse conditions. Our findings conclusively demonstrate that applying exogenous Pro and GB to leaves effectively counteracts the salt-induced decrease in SPAD value. This outcome is due to the osmoprotectant properties of Pro and GB, which act as antitranspirants, allowing the plant to access more water over a prolonged period. Consequently, photosynthesis is facilitated, contributing to the observed reduction in stress-related effects on the SPAD value (Dawood et al., 2021). Relative water content (RWC) serves as a reliable indicator of plant stress tolerance, offering valuable insights into their resilience and adaptability to challenging conditions. Salinity stress induces osmotic stress, which has been shown to impact plants' RWC (Mushtaq et al., 2023). Accumulation of phytotoxic ions such as Na+ and Cl− in leaves disrupts nutrient balance, significantly reducing the concentrations of essential elements like K+ and Ca2+. This disruption in ion equilibrium negatively affects plant water relations, leading to decreased RWC and leaf water potential (Acosta-Motos et al., 2017). In this study observed that increased salt concentrations resulted in a decrease in RWC. However, foliar application of Pro and GB reinstated RWC, corroborating earlier findings by Tang et al. (2015). Salt stress significantly affected the yield-contributing characteristics (e.g., tiller number hill−1) of proso millet. The most significant reduction (41%) occurred under S2 compared to S1, measured at 30, 50 DAS, and during harvest. However, Pro proved more effective than GB in counteracting the negative effects of salt stress on tiller number hill⁻1 under both salt stress doses from 30 DAS to harvest. Salinity severely affects plants by impairing their reproductive capacity through hindrances in photosynthesis and assimilate production, leading to diminished yield and overall productivity (Arif et al., 2020; Shah et al., 2021). In reaction to increased soil salinity, plants initiate expedited water loss from their cells. For proso millet, water is critical for flowering and panicle formation, especially during the reproductive stage. Therefore, water scarcity significantly impacts the plant's reproductive processes (Yuan et al., 2022). 80 Alam & Hasanuzzaman The current study's results demonstrated that salt stress decreased panicle length, filled grain numbers, and 1000-grain weight, correlating with the reduction of proso millet's grain yield. Under salt stress conditions, Pro proved more effective than GB in mitigating negative effects on yield-contributing parameters like tiller number hill−1. Exposure to S1 and S2 resulted in a decrease in panicle length by 22% and 47%, respectively, compared to the control. However, both Pro and GB performed better under S2 than S1, increasing the panicle length by 17 and 11%, respectively (Table 2). Table 2. The number of tillers hill−1 at 30 DAS, 50 DAS, harvest and panicle length of proso millet under salt stress supplemented by Pro and GB. Treatment Number of tiller hill−1 Panicle length (cm) 30 DAS 50 DAS harvest C 1.444±0.040 a 3.989±0.016 a 3.6696±0.1372 a 18.8±0.62 ab Pro 1.455±0.093 a 3.874±0.147 ab 3.6482±0.1401 a 19.3±0.58 a GB 1.479±0.041 a 3.737±0.074 b 3.6397±0.0452 a 18.5±1.05 b S1 1.094±0.030 c 2.964±0.045 e 2.9582±0.0453 c 14.7±0.52 d S1 + Pro 1.234±0.068 b 3.517±0.130 c 3.3957±0.0596 b 16.3±0.32 c S1 + GB 1.220±0.033 b 3.345±0.126 cd 3.0477±0.1162 c 15.6±0.86 c S2 0.851±0.047 e 2.519±0.064 f 2.1923±0.1233 e 11.8±0.24 f S2 + Pro 0.971±0.039 d 3.183±0.130 d 2.6402±0.0991 d 14.2±0.51 d S2 + GB 0.943±0.047 d 2.821±0.109 e 2.6010±0.0950 d 13.3±0.74 e The application of various salt levels had a negative impact on other yield-contributing parameters, such as filled grain panicle−1, unfilled grain panicle−1, 1000-grain weight, and the total yield of proso millet. Exposure to S2 led to a 40% decrease in filled grain panicle−1 compared to the control. Nevertheless, both Pro and GB were more effective under S1 than S2, increasing the number of filled grain panicle−1by 15- and 12%, respectively (Figure 3A). Similarly, there was a significant increase in unfilled grain panicle−1 by 28% under S2 compared to the control. However, the application of supplemental SA and Pro increased the number of unfilled grain panicle−1 under both S1 and S2, with Pro proving more effective than GB (Figure 3B). Fig. 3. The number of filled (A) and unfilled (B) grains panicle−1 of proso millet under salt stress supplemented by salicylic acid and Pro. Here, Pro and GB represent Pro and GB, respectively A significant decrease in 1000-grain weight was observed under S2, with a reduction of 39%, while it was only 23% under S1 compared to the control. Pro demonstrated better efficacy than GB, resulting in a 21% increase in 1000-grain weight under S2 (Figure 4A). Under salt stress conditions, grain yield hill−1 was diminished by 19% and 41% under S1 and S2, respectively, Enhancing salt stress tolerance of proso millet by proline and glycinebetaine 81 compared to the control. However, the supplementation of Pro enhanced the grain yield hill−1 by 19% at S2 compared to S1 (Figure 4B). Fig. 4. The 1000-grain weight (A) and grain yield (B) of proso millet under salt stress supplemented by salicylic acid and Pro. Here, Pro and GB represent Pro and GB, respectively. The results of this experiment underscored the vital role of externally applied Pro and GB in maintaining optimal physiological processes under salt stress. As osmo-protectants, Pro and GB have the potential to reduce excessive water loss from cells, enabling plants to cope with water stress during periods of salt-induced osmotic imbalance. By promoting photosynthesis, they contribute to increased carbohydrate production, which is crucial for improving yield-contributing characteristics such as biomass accumulation and grain filling. Moreover, by mitigating the adverse effects of salinity, these compounds enhance yield- contributing parameters, including biomass production, grain filling, and overall crop productivity. Thus, they play indispensable roles in improving crop performance in saline environments (Tabssum et al., 2018; Shemi et al., 2021). These findings underscore the valuable benefits of Pro and GB in supporting plant growth and productivity in challenging environments. Conclusion The results of this study provide compelling evidence that the individual and combined supplementation of Pro and GB successfully mitigated the adverse effects of salt stress on proso millet. These treatments demonstrated a significant enhancement in the salt tolerance of proso millet. Under salt stress conditions, the exogenous application of Pro and GB resulted in notable improvements in both morphological and yield parameters of proso millet. Moreover, the application of Pro exhibited superior efficacy compared to GB in conferring resistance to different levels of salt stress. These findings highlight the potential of Pro and GB as valuable tools for enhancing salt stress tolerance in proso millet and potentially other crops. Further research and field trials will be required to explore their application in agricultural practices and their long-term effects on crop productivity. Acknowledgements We would like to express our appreciation to the Ministry of Science and Technology, Government of the People's Republic of Bangladesh, for their valuable financial support in enabling the execution of this research work. 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