Effect of selenium biofortification on physiological and antioxidant parameters in lentil (Lens culinaris) plants

Introduction

Can targeted micronutrient enrichment of staple crops bridge the gap between agricultural productivity and nutritional security? Selenium (Se) deficiency affects an estimated one billion people worldwide [1] . Lentil (Lens culinaris Medik.) ranks as the second most consumed pulse globally, yet Se concentrations in its grain vary widely [2] . Biofortification has emerged as a cost-effective strategy to address hidden hunger [3] . At low to moderate concentrations, Se promotes chlorophyll biosynthesis and strengthens antioxidant defense [4] . Plants treated with Se show increased SOD, CAT, and APX activities [5] . However, excessive Se disrupts enzyme function and triggers oxidative stress [6] . Foliar application bypasses variable soil adsorption and allows precise dose control [7] , with sodium selenate preferred for its phloem mobility [8] . Broadley et al. [9] reported foliar Se increased grain Se in wheat without yield penalty. Boldrin et al. [10] observed improved antioxidant status in rice at 2-5 mg/L selenate. Yet dose-dependent changes in chlorophyll metabolism, water relations, and enzymatic antioxidant defense in lentil remain unclear. This research evaluated four foliar Se concentrations on physiological and antioxidant parameters in lentil under controlled conditions.

Materials and Methods

Study area and environmental conditions

The experiment was conducted at the University of Florida greenhouse, Gainesville (29.65° N, 82.32° W) from January to April 2022. Day/night temperatures were maintained at 26/18±2 °C with a 14-hour photoperiod and 55-65% relative humidity. PAR at canopy level was 410-480 µmol m⁻² s⁻¹ [13] . Soil was Millhopper fine sand from the university research farm.

Materials

Certified lentil seeds (cv. Indianhead) from the USDA Pulse Crop Germplasm Collection were surface-sterilized with 0.1% sodium hypochlorite and inoculated with Rhizobium leguminosarum bv. viciae. Five seeds were sown per 8 L pot filled with Millhopper fine sand amended with 2 g NPK (15-15-15), then thinned to three per pot. Five treatments of foliar sodium selenate (Na₂SeO₄, 98.5% purity) at 0, 2, 4, 6, and 8 mg Se/L were replicated four times in a completely randomized design.

Methods

Foliar Se was sprayed at 30 and 55 days after sowing (DAS) with 0.05% Tween-20 as surfactant. Leaf samples were collected at 30, 55, 75, and 95 DAS. Total chlorophyll was extracted in 80% acetone following Arnon's method [14] . Relative water content was determined gravimetrically. SOD activity was assayed by NBT photoreduction [5] , CAT by H₂O₂ decomposition at 240 nm, and APX by ascorbate oxidation at 290 nm. MDA was quantified by the TBARS method. Grain Se was analyzed by ICP-MS. Data were subjected to one-way ANOVA (SAS 9.4), with means separated by Tukey's HSD at p≤0.05.

Results

Comprehensive interpretation

Se biofortification produced clear dose-dependent responses. Total chlorophyll increased from 2.14 mg/g FW (control) to 2.68 mg/g FW at 4 mg/L Se at flowering a 25.2% increase (p<0.01). Antioxidant enzymes followed a bell-shaped curve (Figure 1): SOD peaked at 52.3 U/mg protein under 4 mg/L Se versus 34.2 in the control. MDA was lowest at 4 mg/L (3.14 nmol/g FW) and rose to 5.87 at 8 mg/L, indicating membrane damage (Table 2). Se-treated plants maintained higher RWC throughout reproductive stages (Figure 2). Grain Se reached 0.87 mg/kg at 4 mg/L.

Discussion

The bell-shaped response to increasing Se aligns with the hormesis model. At moderate doses, Se stimulated the ascorbate-glutathione cycle, upregulating SOD, CAT, and APX. This matches Hartikainen et al. [4] , who observed sub-toxic Se acted as a pro-oxidant trigger in ryegrass. The chlorophyll increase at 4 mg/L is consistent with selenium's role in protecting chloroplast membranes from ROS degradation [6] . By lowering lipid peroxidation, Se preserved thylakoid membrane integrity. Boldrin et al. [10] reported a comparable 22% chlorophyll increase in rice. Declining enzyme activities at 8 mg/L and concurrent MDA rise confirm a toxicity threshold through selenoamino acid incorporation [6] . Grain Se at 0.87 mg/kg with 4 mg/L provides meaningful dietary contribution assuming 50 g daily consumption, this supplies about 43.5 µg Se, near the WHO recommended 55 µg [15] .

Conclusion

Foliar selenium at 4 mg/L sodium selenate was the most effective treatment for improving physiological performance and antioxidant defense in lentil. This concentration maximized chlorophyll content, relative water content, and SOD, CAT, and APX activities without oxidative damage. Grain Se reached 0.87 mg/kg, potentially supplying 79% of the daily recommended intake at typical consumption rates. At 8 mg/L, toxicity was evident through declining enzyme activities and rising MDA. The two-spray foliar regime was simple and accessible to smallholder farmers. Future wor should validate these findings under field conditions across multiple seasons and soil types.

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