Morphological and biochemical evaluation of foxtail millet (Setaria italica) varities under moisture stress conditions

Introduction

Millets are a group of small seeded cereal grasses which do not shatter easily at maturity. The French word "mille," meaning thousand, is the origin of the word "millet" which suggests that a handful of millet contains thousands of grains (Taylor, 2019) [1] . Because of their long history of cultivation and consistent yield, these are frequently referred to as "heritage crops." Often referred to as "coarse cereals" these rainfed crops do best in arid and semi-arid regions of the world and are well-suited to marginal soil fertility and moisture conditions. With their many applications as food, feed, fodder, biofuels, and brewing, millets are the foundation of dryland agriculture. They benefit not only consumers but also farmers and the environment so these are referred to as "smart foods." The Government of India (GoI) took a step forward and submitted a proposal to the United Nations (UN) to declare 2018 as the National Year of Millets in an effort to promote millets and boost domestic production. quire little to no purchased inputs. The Food and Agriculture Organization (FAO) declared the year 2023 the "International Year of Millets" after carefully considering India's position. As we face challenges like climate change and rising rates of obesity, millets offer a path toward a more sustainable and nutritious food system. Because of their resilience, millets have been dubbed "the ultimate famine insurance". Some types can remain edible for decades if stored properly. Foxtail millet (Setaria italica) is a widely grown crop in arid and semi-arid regions for its short growth cycle and drought tolerance and is mainly grown as rainfed crop on marginal soils. It is cultivated on about 2.5 million ha worldwide, with 85-90% of the area in Asia, particularly China and India. In India its cultivation is predominantly in Andhra Pradesh, Karnataka and Tamil Nadu with some pockets in north-eastern states under rainfed and shifting cultivation systems (Brunda et al., 2014) [2] . The highest productivity was recorded in the year 2022 with production of 3.6 lakh tons from 1.52 lakh ha at 2363 kg ha -1 in Andhra Pradesh (NABARD, 2023). Foxtail millet is known for nutritional and climate resilient properties, but has always received little attention due to the dominance of rice and wheat, low productivity and processing constraints (Upadhayaya et al., 2018) [3] . Popularly called Koni dhan in Assam. It is grown mainly in the tribal and rainfed upland areas, mainly in the Brahmaputra valley and adjoining hill districts. Its adaptability to subtropical conditions makes it a promising climate resilient crop.

Millets are often referred to as "nutri-cereals" or "miracle grains" because of their higher nutritional and functional values compared to the major cereals (FAO, 2021; Saini et al., 2021) [4] . Foxtail millet is a good source of protein, dietary fiber, carbohydrates, minerals, vitamins and bioactive compounds such as phenolic and phytic acids (Kumar et al., 2016; Gowda et al., 2022) [6,5] . Its grains have 12-16% protein and 4-8% fat and appreciable quantities of iron, zinc, calcium and essential amino acids (Sachdev et al., 2021; Yang et al., 2022) [7,8] . Foxtail millet is a high-fibre, low-glycemic index, glutenfree grain with antioxidant activities and is useful for diabetes, cardiovascular health, digestive system, and weight management (Mohammed et al., 2009; Zhang et al., 2024) [9,10] . Its extensive nutritional profile also highlights its potential as a biofortification and climate resilient food crop (Hariprasanna, 2023) [11] . Climate change is increasing temperature variability, changing rainfall patterns, and intensifying water scarcity and extreme weather, which are major challenges to agricultural production (IPCC, 2021; Hoekstra et al., 2012) [12] . Drought stress causes loss in yield by causing stomatal closure, reduced CO 2 assimilation, oxidative stress and disruption of photosynthetic and metabolic processes. These effects are alleviated by plants through the production of osmolytes such as proline, glycine betaine and trehalose that help in osmotic adjustment and cell protection. In addition, high temperature stress (>32-35 °C) disrupts physiological processes and may lower crop productivity by 10-40% during sensitive stages (Chakraborty et al., 2025) [13] . The climate-resilient nature of foxtail millet growth, yield and grain quality is under threat due to drought and unpredictable rainfall patterns especially in rainfed systems. These stresses are of concern to the rainfed and tribal farming communities in Assam and could be exacerbated under combined stress conditions (Swain et al., 2024; Terfa et al., 2025) [14,15] . Water stress during vegetative growth reduces the tillering, but drought at reproductive phase increases the number of unfilled grains and significantly reduces the yield (Terfa et al., 2025) [15] . Therefore, understanding these responses is important for developing climate-resilient foxtail millet production systems. Foxtail millet is generally cultivated under rainfed circumstances by marginal farmers and can incur large yield losses under drought stress. Although it demonstrates C 4 metabolism, deep root system and antioxidant and osmolyte defenses provide great climate tolerance. Its biochemical reactions to drought particularly in local Assam landraces are little characterized. Therefore, the present study was undertaken to examine the physiological and biochemical responses of foxtail millet to drought stress addressing an essential research need under changing climatic conditions along with application of Salicylic Acid for its mitigation.

Material and methods

Plant material, experimental site and drought stress

Two varieties of foxtail millet (Setaria italica (L.) P. Beauv.) namely AAU GSG CAWN-1 (white seeded) and Gossaigaon Local (red seeded) procured from Assam Agricultural University, Zonal Research Station, Gossaigaon were tested under drought stress conditions. The experiment was carried out at the ICR Farm, Assam Agricultural University, Jorhat (26°41′N, 94°19′E; 86.6 m amsl) in 2024 in a greenhouse. In the experiment temperature and relative humidity varied between 12.5-36.5 °C and 69.84-94.36 %, respectively. Irrigation was scheduled based on crop evapotranspiration (ET c ) calculated as 𝐸𝑇 c = 𝐾 c × 𝐸𝑇 o , where K c is the crop coefficient and ET o is reference evapotranspiration.

Details of treatment

Five treatments were applied: T 1 , normal irrigation (ET c ); T 2 , 50% irrigation; T 3 , no irrigation; T 4 , 50% irrigation + SA; and T 5 , no irrigation + SA, with two varieties and three replications.

Seed priming and application of SA

Seeds were primed in 1 mM salicylic acid (SA) and control seeds were soaked in distilled water for same duration and shade dried before sowing. Foliar application of 1 mM SA was performed at 40 and 60 DAS as per Yadav et al. (2020) [16] .

Morphological and germination characteristics

Plant height was measured at 40, 80 and 120 days after sowing (DAS), while tillers and panicles plant -1 were recorded at 40 and 80 DAS. Leaf area was calculated as length x maximum width x 0.75.

Chemical analysis

Chlorophyll a and b were determined by the DMSO method of Hiscox et al. (1979) [17] . Leaf samples were incubated at 65 °C for 4 h in DMSO and absorbance was measured at 645 and 663 nm. Contents were calculated according to Arnon (1949) [18] and expressed as mg g -1 FW. Proline was estimated by method of Bates et al. (1973) [19] using 3% sulfosalicylic acid, acid ninhydrin and glacial acetic acid. Absorbance was read at 520 nm after extraction with toluene and content was expressed as μmol g -1 FW. MDA was measured as an index of lipid peroxidation according to Heath et al. (1968) [20] . Leaf extracts were treated with TCA-TBA and heated at 95 °C for 30 min and absorbance was measured at 532 nm corrected at 600 nm. MDA content expressed as µmol g -1 FW. Crude fat was determined by Soxhlet extraction with petroleum ether (40-60 °C) for 6-8 h (AOAC 920.39). After extraction the solvent was removed and the extracted lipid in the flask was dried at 100-105 °C for 30 min, cooled in a desiccator and weighed. The crude fat content was calculated from the increase in flask weight and expressed as a percentage of the sample weight. Total carbohydrate was determined by the anthrone method of Hedge et al. (1962) [21] . The finely ground sample (100 mg) was hydrolyzed with 5 mL 2.5 N HCl in boiling water bath for 3 h, cooled, neutralized with sodium carbonate, made up to 100 mL and centrifuged at 10,000 rpm for 15 min. An aliquot (0.5 mL) was mixed with 4 mL of anthrone reagent (200 mg anthrone in 100 mL ice-cold 95% H 2 SO 4 ) and heated in a boiling water bath for 5 min. Absorbance was measured at 630 nm with glucose standards (10-100 µg) and carbohydrate content was expressed as percentage on dry weight. Crude protein was analyzed by Kjeldahl method (AOAC 979.09) 0.5-1.0 g sample was digested with concentrated H 2 SO 4 and Kjeldahl catalyst at 380-420 °C until clear, followed by dilution and distillation with 40% NaOH. The liberated ammonia was absorbed in 4% boric acid containing mixed indicator and titrated against 0.1 N HCl. Nitrogen content was calculated from sample and blank titre values. Crude protein was calculated by multiplying the nitrogen content by 6.25 and expressed in percentage on dry-weight basis.

Result and discussion morphological characteristics under moisture stress

Moisture stress significantly decreased the growth and yield related morphological traits of both the foxtail millet varieties while salicylic acid (SA) alleviated these effects to a certain degree. Maximum plant height was recorded in T 1 (normal irrigation) at 40, 80 and 120 DAS (52.72, 142.80 and 144.80 cm in V 1 ; 66.30, 156.60 and 160.60 cm in V 2 , respectively). Maximum reduction in plant height was observed in T 3 (complete withdrawal of irrigation) (37.83, 106.00 and 110.00 cm in V 1 ; 27.21, 95.60 and 104.21 cm in V 2 ). The application of SA improved the plant height under stress, particularly at later stages, where T 3 +SA achieved 136.80 cm in V 1 and 148.25 cm in V 2 at 120 DAS. Similar trend was observed in the production of tiller and panicle which was decreased under moisture stress and lowest values were obtained in T 3 . The application of SA improved their formation especially at 80 DAS. At 80 DAS, control plants recorded 6.10 and 6.24 tillers and 9.07 and 8.32 panicles plant -1 in V 1 and V 2 , respectively, as against 4.12 and 4.85 tillers and 5.02 and 4.75 panicles plant -1 under T 3 . SA increased these values to 5.64 and 5.26 tillers and 5.72 and 6.29 panicles plant -1 , respectively. Leaf area was also significantly reduced by moisture stress, particularly under T 3 , from 487.40 and 401.00 cm 2 in control plants to 184.00 and 213.50 cm 2 at 80 DAS in V 1 and V 2 , respectively. SA application partially recovered the leaf expansion; T 3 +SA had 241.30 and 296.40 cm 2 . In general, water stress affected cell growth, photosynthetic assimilation and vegetative/reproductive development, while SA was involved in plant growth and leaf development under water deficit in agreement with previous reports (Yadav et [16,22] .

Biochemical characteristics under moisture stress

The contents of chlorophyll a and b were decreased under moisture stress in both the varieties of foxtail millet, and the maximum reduction was observed under complete irrigation withdrawal (T 3 ). These effects were somewhat alleviated by salicylic acid (SA). Chlorophyll a decreased from 1.61 and 1.54 mg g -1 FW under control to 1.41 and 1.28 mg g -1 FW under T 3 in V 1 and V 2 , respectively, while chlorophyll b decreased from 0.74 and 0.71 to 0.51 and 0.59 mg g -1 FW at 40 DAS, respectively. The control plants at 80 DAS had 3.67 and 2.73 mg g -1 FW chlorophyll a and 1.74 and 1.71 mg g -1 FW chlorophyll b, respectively, which were reduced to 1.06 and 0.92 and 0.51 and 0.59 mg g -1 FW, respectively, in T 3 . SA application enhanced pigment retention under stress, T 3 +SA led to increase in chlorophyll a to 2.30 and 2.36 mg g -1 FW and chlorophyll b to 1.48 and 1.60 mg g -1 FW at 80 DAS in V 1 and V 2 respectively. The reduction in chlorophyll due to moisture stress could be either due to reduced biosynthesis or enhanced degradation of chlorophyll or oxidative damages to chloroplasts. SA may regulate the biosynthesis of chlorophyll as well as help protect the photosynthetic machinery through improved antioxidant defense and reduced ROS induced damages. Similar responses were also observed in foxtail millet and other cereals under drought stress (Yadav et [16,22] . The proline and malondialdehyde (MDA) contents were significantly affected by moisture stress in both foxtail millet varieties, and the most significant effects were observed under complete irrigation withdrawal (T 3 ), whereas the effects were mitigated by salicylic acid (SA). Proline increased from 1.00 and 0.74 mg g -1 FW under control to 1.76 and 1.16 mg g -1 FW under T 3 in V 1 and V 2 respectively at 40 DAS and further increased at 80 DAS to 3.87 and 3.56 mg g -1 FW compared to 2.49 and 1.45 mg g -1 FW in controls. SA reduced the accumulation of proline due to stress. T 3 + SA recorded 2.14 and 2.24 mg g -1 FW at 80 DAS. In contrast, MDA was increased significantly under drought from 20.20 and 20.86 µmol g -1 FW in control to 80.30 and 82.29 µmol g -1 FW under T 3 at 40 and 80 DAS, respectively. SA application reduced MDA accumulation especially in severe stress condition, where T 3 +SA showed 54.30 and 50. Moisture stress decreased crude fat, total carbohydrate and crude protein contents of leaves of both foxtail millet varieties, while salicylic acid (SA) partially alleviated these reductions. The control plants at 40 DAS showed the highest crude fat (4.87 and 4.43 %), total carbohydrate (64.50 and 68.02 %) and crude protein (10.91 and 11.15 %) in V 1 and V 2 , respectively, whereas complete irrigation withdrawal (T 3 ) decreased these values to 3.98 and 3.80 %, 45.85 and 60.66 %, and 3.80 and 9.50 %, respectively. Reductions were more severe at 80 DAS, particularly under T 3 , where crude fat was reduced to 1.21 and 2.12 %, carbohydrate to 48.45 and 48.15 % and protein to 10.56 and 10.25 % in V 1 and V 2 , respectively as compared with higher values under control conditions. SA application partially restored these biochemical constituents. At 80 DAS, T 3 +SA increased crude fat to 3.55 and 2.22, carbohydrate to 38.15 and 50.12 and protein to 11.25 and 11.98 % in V 1 and V 2 respectively. Under moisture stress the reductions may be due to impaired photosynthetic carbon assimilation, reduced nutrient uptake and disruption of lipid and protein metabolism. SA may help to maintain metabolic activity through improved stress tolerance. Similar effects of drought stress on nutritional and metabolic constituents have been reported in millets and cereals (Harish et al., 2024).

Conclusion

The study revealed significant effect of moisture stress on morphological and biochemical performance of two foxtail millet varieties. Complete withdrawal of irrigation resulted in highest reductions of plant height, tillering, panicle formation, leaf area, chlorophyll, carbohydrate, crude fat and protein contents. Conversely, proline and MDA increased under severe moisture stress indicating osmotic adjustment and oxidative membrane damage. Application of salicylic acid partially alleviated these detrimental effects by improving plant growth, leaf expansion and chlorophyll retention and reducing MDA accumulation. The responses observed in AAU GSG CAWN-1 and Gossaigaon Local suggest that SA can help in the maintenance of physiological and biochemical stability of foxtail millet under moisture-deficit conditions.

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