Fulvic Acid Enhances Oat Growth and Grain Yield Under Drought Deficit by Regulating Ascorbate–Glutathione Cycle, Chlorophyll Synthesis, and Carbon–Assimilation Ability

Abstract

Drought deficit inhibits oat growth and yield. Fulvic acid (FA) can enhance plant stress tolerance, but its effects on regulating the ascorbate–glutathione cycle, chlorophyll synthesis, and carbon–assimilation ability remain unclear. Therefore, this study aimed to elucidate the physiological mechanisms of the FA regulation of drought tolerance in oats and its relationship with growth and yield using the drought-resistant variety Yanke 2 and the drought-sensitive variety Bayou 9. The effects of FA on growth and yield, the antioxidant system, chlorophyll synthesis, and carbon–assimilation capacity of oats under drought stress were investigated by systematically assessing changes in morphogenesis, ascorbate–glutathione cycle, chlorophyll and its intermediates, carbon–assimilation enzyme activities, and carbohydrate metabolism. The results showed that under drought stress, FA treatment significantly promoted oat growth (leaf area, dry matter) and yield, elevated glutathione peroxidase, ascorbate peroxidase, glutathione reductase, and dehydroascorbate reductase activities, reduced ascorbic acid, and reduced glutathione content. In addition, FA increased chlorophyll, as well as magnesium protoporphyrin IX, protoporphyrin IX, and protochlorophyllin acid ester content, enhanced 1,5-bisphosphate ribulose carboxylase, 1,5-bisphosphate ribulose carboxylase enzyme, 1,7-bisphosphate sestamibiose heptulose esterase, 1,6-bisphosphate fructose aldolase, sucrose synthase, sucrose phosphate synthase, acid invertase, and neutral invertase activities, and increased sucrose, glucose, and fructose content. Overall, fulvic acid (FA) alleviates drought-induced damage in oats by enhancing the ascorbate–glutathione cycle, promoting chlorophyll biosynthesis, and improving carbon assimilation and carbohydrate metabolism. The drought-sensitive variety (Yanke 2) was more effective in application compared to the drought-resistant variety (Bayou 9). This research provides valuable insight into its potential as a biostimulant under abiotic stress.

1. Introduction

Oats (Avena sativa L.), an annual cereal, are an important global food crop [1]. Oats have attracted attention for their rich content in various minerals, proteins, dietary fiber, vitamins, and antioxidants. They are widely used as medicinal and food ingredients, offering benefits such as weight loss, lowering blood sugar and lipid levels, improving cardiovascular health, and enhancing immunity [2,3]. The market demand for oats is rising annually as people’s focus on health and nutrition continues to grow [4]. The development of the oat industry can not only meet the market demand for healthy food but also promote agricultural economic growth and improve the nutritional status of the population [5]. Therefore, increasing support and investment in the oat industry has significant economic and social implications. Oats are widely distributed globally, and according to the Food and Agriculture Organization of the United Nations, the global cereal crop oats is ranked sixth in terms of area planted [6]. China is one of the world’s largest producers and consumers of oats, as well as the main production area for large-grain oats, mainly in the arid and semi-arid regions of North China, Northwest China, and Southwest China. These areas are characterized by harsh natural conditions and low rainfall, with drought and water scarcity being the most important factors limiting oat growth and yield [7,8]. The study showed that there were significant differences in the period of occurrence, duration, and intensity of drought on yield effects. Terminal droughts occurring between flowering and filling periods caused significantly higher yield losses than droughts at other fertility stages [9]. Light and severe droughts during flowering can lead to reductions in wheat yields of 8–19% and 11–51%, respectively [10].

Drought stress reduces the growth and productivity of plants by affecting their stomatal opening, endogenous hormones, osmotic substances, and antioxidant systems [11]. Photosynthesis is the first physiological process to be affected by drought stress. Drought decreases CO₂ assimilation and photosystem II activity in plants, blocking electron transfer, accelerating chlorophyll degradation, reducing normal plant metabolism, inducing early plant failure, and leading to a decrease in yield [12]. Sukhova et al. [13] found that under water-deficient conditions, plants reduce water loss by closing their stomata, limiting CO₂ entry into the leaves, which directly affects the carbon–assimilation process. At the same time, it also leads to the ineffective transfer of excess electrons generated in the light reaction, driving the excess generation of reactive oxygen species (ROS), which inhibits its antioxidant defense system, resulting in increased oxidative damage, and in order to mitigate this damage, its plants produce a large number of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), etc.) or non-enzymatic antioxidants (e.g., ascorbate, proline, and sugar) to help the plants under drought stress to maintain the stability of cell structure and function under drought stress [14]. It has been shown that drought stress stimulates the ascorbic acid defense system in wheat leaves to increase the activities of APX, which scavenges H₂O₂, and Monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) enzymes, which accelerate the regeneration of the AsA cycle, thereby maintaining leaf ascorbic acid levels and alleviating drought damage to the plant [15]. Under drought stress, plants also enhance drought resistance by synthesizing large amounts of carbohydrates and regulating the osmotic potential of cells [16]. Although plants have evolved complex adaptive mechanisms to cope with water scarcity, these mechanisms cannot mitigate the damage caused by drought. Early studies have shown that plant growth regulators play a central role in modulating the signaling and physiological and biochemical pathways by which plants respond to adversity stress [17,18,19]. Therefore, foliar spraying of these compounds is an important agricultural management practice that can effectively enhance crop defense and adaptation to environmental stresses, such as drought.

Fulvic acid (FA) is a brownish-red powdery substance easily soluble in water, containing carboxyl groups, phenolic hydroxyl groups, alcohol hydroxyl groups, and other oxygen-containing functional groups, belonging to the low-molecular-weight natural organic polymers, easily absorbed by the plant and utilized [20]. FA, as a plant growth regulator, is involved in regulating multiple biological processes of plant development [21], including improving seed vigor [22], promoting growth [23,24], enhancing photosynthetic capacity [25], and regulating oxidative damage [26]. Numerous studies have confirmed that FA can effectively enhance plant adaptation to abiotic stresses. Specifically, FA activates the activities of SOD, CAT, peroxidase (POD), and polyphenol oxidase (PPO) to reduce oxidative stress in plants [27,28]. Studies have shown that FA improves crop tolerance to heavy metal stress by enhancing the antioxidant defense system, promoting photosynthetic pigment synthesis, and regulating carbon and nitrogen metabolism [27,29,30]. Jarošová et al. [31] further reported that humic acid protects barley from NaCl stress by limiting Na⁺ uptake and positively regulating the levels of metabolites (e.g., butyric acid, alanine, proline, ascorbic acid, glutathione). In addition, FA improves net photosynthesis, increases chlorophyll content, and maintains chloroplast ultrastructure and electron transfer efficiency, thereby mitigating the adverse effects of drought on cucumber photosynthesis [32]. Fang et al. [33] also demonstrated that FA treatment improves the photosynthetic and chlorophyll fluorescence parameters and maintains the integrity of chloroplasts in peonies.

All of the above studies confirmed that FA plays a significant role in alleviating abiotic stress in plants, and its mechanism mainly enhances plant stress tolerance by regulating antioxidant capacity, increasing chlorophyll content, and improving photosynthetic rate, fluorescence parameters, and carbon and nitrogen metabolism. However, there is still a need to further explore how FA affects chlorophyll synthesis and how it regulates carbon–assimilation processes. Our previous study found that FA promoted oat growth under drought stress by attenuating membrane lipid peroxidation, modulating the antioxidant system, phenylpropanoid biosynthesis, and glutathione metabolic pathways in oat leaves [26]. These studies have mainly investigated the mechanisms by which FA mitigates the adverse effects of drought on oats from antioxidant and growth perspectives, without integrating the physiological effects with detailed biochemical parameters, especially in varieties with different responses to drought. Based on the available evidence, we hypothesized that (1) FA application enhances ascorbate–glutathione cycle activity in drought stressed oats; (2) FA maintains chlorophyll synthesis and carbon–assimilation processes under drought conditions; (3) FA-mediated improvements in these physiological processes correlate with increased oat growth and yield. Therefore, the present study focused on the effects of FA on ascorbate–glutathione cycling, chlorophyll synthesis, and carbon assimilation in oat under drought stress, aiming to elucidate the mechanism of FA-induced drought tolerance in different oat cultivars at the physiological level. The results will provide a new theoretical basis for further revealing the role of FA in mitigating drought stress and fill the research gap on the mechanism of FA in enhancing plant drought resistance through the regulation of photosynthesis.

2. Materials and Methods

2.1. Plant Material and Experimental Treatments

In this study, the drought-resistant variety ‘Yanke 2’, provided by the Academy of Agricultural and Animal husbandry sciences of the Inner Mongolia Autonomous region and the moisture-sensitive variety ‘Bayou 9’, selected by the Alpine Crops Research Institute of Hebei Province were used as test materials. Thirty seeds were planted in plastic pots measuring 25 cm in diameter and 18 cm in depth, each filled with approximately 3 kg of a soil mixture composed of soil, vermiculite, and peat soil in a 1:1:1 volume ratio. The basic properties of the soil are shown in

Table 1. Basic soil property data.

PH	Total Nitrogen (g kg−1)	Available Phosphorus (mg kg−1)	Available Potassium (mg kg−1)
7.32	1.32	18.5	145.2

. At the third leaf stage, the number of seedlings per pot was reduced to 20. The seedlings were cultivated in a controlled greenhouse at Inner Mongolia Agricultural University in Hohhot, China (40.818° N, 111.725° E) in 2022, under conditions of 20 ± 2 °C, a 16 h light and 8 h dark photoperiod, and a light intensity of 2000 lux. Before the jointing stage, soil moisture in all pots was maintained at 75% of the field capacity by weighing the pots every two days and adding water as necessary. Drought stress was initiated at the jointing stage (32 days after planting) by maintaining soil water content at 45%, and this stress condition was maintained until plants reached maturity (113 days after planting).

The trial was a randomized complete block design. All experimental treatments were set up as follows: (1) distilled water was sprayed to 75% field capacity (WW); (2) distilled water was sprayed and drought stress was induced (DS); (3) FA was sprayed and drought stress was induced (DF). Three replicates (pots) were set up for each treatment with four pots per replicate, totaling seventy-two pots. FA and distilled water were uniformly applied to oat leaves through precision foliar spraying using a handheld sprayer from 9 a.m. to 11 a.m. during the jointing, heading, and filling, with an application amount of about 15 mL per pot. The concentration of FA used in this study was 600 mg/L, based on our previous research results [34]. After 7 days of FA treatment during the filling period, growth indicators were determined for each replicate, while flag leaf samples were collected, rapidly frozen in liquid nitrogen, and stored in an ultra-low-temperature refrigerator at −80 °C for subsequent determination and analysis.

2.2. Growth Indicators and Yield

The flag leaves of oat plants were selected, and the length and width of the leaf blades were measured with a straightedge to calculate the leaf area using the aspect coefficient method with the following formula [35]:

Leaf area = leaf length × leaf width × 0.73 (empirical coefficient)

(1)

Flag leaves of 20 oats were selected for each replication, packed into paper bags, and placed in an oven, first killed at 105 °C for 20 min, and subsequently dried at 80 °C until constant weight, which is the dry matter of the leaves.

Twenty oat plants were selected for each replication, and after removing the roots, they were put into separate paper bags, placed in an oven, first killed at 105 °C for 20 min, and subsequently dried at 80 °C until constant weight, which is the aboveground dry matter mass.

At maturity, potted plants of uniform growth status were selected for each replication, and the weight of seeds per pot was determined as the yield.

2.3. Ascorbic Acid-Glutathione Cycle

Plant samples were rapidly frozen in liquid nitrogen immediately after collection and stored at −80 °C until analyzed (within a month). The activities of glutathione peroxidase (GPX), ascorbate peroxidase (APX), glutathione reductase (GR), and dehydroascorbate reductase (DHAR), as well as the contents of oxidized ascorbate (DHA), reduced ascorbate (AsA), reduced glutathione (GSH), and oxidized glutathione (GSSG), were determined by following the instructions of the kits from Shanghai Keshun Science and Biotechnology Co., Ltd. (Shanghai, China), The links to the official website of the kits and the product numbers, as well as the accuracy and sensitivity of the kits, are shown in Tables S1 and S2. The kit is based on the principle of the enzyme-linked immunosorbent assay (ELISA) of the double antibody one-step sandwich method. In the microtiter plate pre-coated with the target-specific antibody, add the sample (10 μL), diluent (40 μL), standard (50 μL), and horseradish peroxidase (HRP)-labeled detection antibody (100 μL) in order, incubate 60 min at 37 °C and wash the plate 5 times, and then wash the plate under the condition of avoiding light for 15 min. The plate was washed 5 times after incubation at 37 °C for 60 min, and then the color was developed by TMB for 15 min under conditions of light-avoidance. The absorbance (OD) was measured at 450 nm within 15 min after the addition of the termination solution, and the actual concentration was converted according to the standard curve (R ≥ 0.99). All operations were performed under strict time, liquid addition sequence, and temperature control.

2.4. Chlorophyll Content

The ethanol extraction method was used [36]. Take oat samples, wash them with distilled water, dry them, and then chop and mix. We took 0.20 g and placed it in a 25 mL volumetric flask, filled it to volume with 95% ethanol, and let it extract in the dark until the leaves turned completely white. Finally, use 95% ethanol as a control and measure the absorbance at 649 nm and 665 nm using a spectrophotometer to calculate the contents of chlorophyll a, chlorophyll b, and total chlorophyll with the specific formulas as follows:

Chlorophyll a Concentration: Chl a = 13.95 × OD₆₆₅ − 6.88 × OD₆₄₉

(2)

Chlorophyll b Concentration: Chl b = 24.96 × OD₆₄₉ − 7.32 × OD₆₆₅

(3)

Total Chlorophyll Content: Total Chlorophyll = Chl a + Chl b

(4)

Photosynthetic Pigment Content (mg·g⁻¹ FW) = (Chl × V × N)/W

(5)

where OD is optical density at specified wavelength, Chl is the pigment concentration, V is the volume of extract (mL), N is the number of dilutions, and W is the fresh weight of the sample (g).

Magnesium protoporphyrin IX (Mg-Proto), protoporphyrin IX (Proto IX), and protochlorophyllide (Pchlide) were all determined by strictly following the methods of the kits of Shanghai Keshun Science and Biotechnology Co., Ltd. These were based on the double-antibody, one-step sandwich assay (ELISA), and the official website of the kits are linked to the product numbers, as well as the accuracy and sensitivity values in Tables S1 and S2. The key operation steps are as follows: equilibrate the reagents at room temperature for 20 min before use, add samples (10 μL), standards (50 μL), and HRP-labeled detection antibody (100 μL) to the microtiter plate pre-coated with antibodies against the targets (Mg-Proto, Proto IX, Pchlide) in sequence, incubate at 37 °C, and then wash the plate thoroughly 5 times. The color was developed by TMB for 15 min under light-avoiding conditions, and the OD value was measured at 450 nm within 15 min after termination. Sample concentration was calculated by the standard curve.

2.5. Carbon Assimilative Enzyme Activity

The leaves and extraction buffer (1:2 ratio) were added to the mortar and pestle to make a homogenate under ice-water bath conditions and then centrifuged at 4 °C and 10,000× g for 15 min. The supernatant was collected, followed by immediate determination of 1,5-bisphosphate ribulose carboxylesterase/oxygenase (Rubisco) activity, 1,5-bisphosphate ribulose carboxylase/oxygenase activase (RCA) activity, 1,7-bisphosphate sestamibiase (SBPase) activity, 1,6-bisphosphate fructose aldolase (FBA) activity, and transketolase (TK) activity were determined immediately following the kit instructions (Shanghai Keshun Science and Biotechnology Co., Ltd., Shanghai, China). Furthermore, the official website of the kits was linked to the product number, and the accuracy and sensitivity values are shown in Tables S1 and S2. The key operation steps are as follows: add the sample (10 μL), standard (50 μL), and HRP-labeled detection antibody (100 μL) to the wells pre-coated with the antibody against the target enzyme, incubate at 37 °C for 60 min, repeat the plate-washing 5 times, then add the TMB substrate (50 μL each of A and B), incubate at 37 °C for 15 min, add 50 μL of termination solution, measure the OD value of each well at 450 nm within 15 min, and calculate the sample concentration by standard curve.

2.6. Carbon Metabolism-Related Enzyme Activities

The sucrose phosphate synthase (SS), sucrose synthase (SPS), acidic invertase (AI), and neutral inverse (NI) activities and sucrose, fructose, and glucose contents were determined in strict accordance with the method of kits from Shanghai Keshun Science and Biotechnology Co., Ltd. (Shanghai, China). The official website link of the kit with the product item number as well as the accuracy and sensitivity are in Tables S1 and S2. The kits were designed based on the principle of the double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The core process is as follows: dilute the sample 5 times (10 μL sample and 40 μL dilution) and sequentially add the standard (50 μL) HRP-labeled detection antibody (100 μL) to the pre-coated microtiter plate with the antibody to the target enzyme, incubate at 37 °C for 60 min, and then wash thoroughly. In total, 50 μL each of the substrates A/B was added to each well, and then the color was developed for 15 min at 37 °C away from light. The reaction was terminated with 50 μL of termination solution, and the OD value at 450 nm was measured within 15 min. The standard curve was plotted with the concentration of the standard as the horizontal coordinate and the OD value as the vertical coordinate, and the actual concentration of the sample was converted according to the linear equation.

2.7. Statistical Analysis

Microsoft Excel 2019 (Microsoft Corp., Redmond, WA, USA) was used for data organization and analysis. Data were tested for normal distribution (Shapiro–Wilk) and homogeneity of variance (Levene) using IBM SPSS Statistics 25 (IBM Corp., Armonk, NY, USA) [37]. The main effects and their interactions were examined using a two-factor analysis of variance model (variety × moisture treatment) (F-test, α = 0.05); One-way ANOVA and Duncan’s multiple range test (DMRT) (α = 0.05) were used to resolve the significance in different treatments for the same variety; inter-variety comparisons were performed by Student’s t-test, where * denotes the level of significance at p < 0.05 and ** denotes the level of significance at p < 0.01. The results were plotted and analyzed using Origin 2024 (OriginLab Corp., Northampton, MA, USA) [38]. Hierarchical cluster analysis was performed for each indicator on the Ouyi Cloud platform (https://cloud.oebiotech.com/) accessed on 20th March 2025. In addition, the combined graphics were created using Adobe Illustrator 15.0.0 (Adobe Inc., San Jose, CA, USA).

3. Results

3.1. Effect of FA on Growth and Yield of Oats Under Drought Stress

Two-way ANOVA showed that moisture treatments had highly significant main effects on oat growth and yield indices, variety type significantly affected leaf area, above-ground dry matter, and yield, while variety–moisture treatment interactions specifically regulated yield (Table S3). The leaf area, leaf dry matter, aboveground dry matter, and yield of both oat varieties were significantly reduced under drought stress (DS) compared with WW, with Yanke 2 having a lower rate of decline than Bayou 9 (Figure 1a–d). However, FA spraying significantly alleviated the inhibitory effects of drought stress on oat growth and yield. The FA treatment (DF) restored 12.7%, 15.1%, 17.5%, and 15.2% of the drought-induced losses in leaf area, leaf dry matter, aboveground dry matter, and yield, respectively, in Yanke 2, whereas the restoration was higher in Bayou 9 (14.9%, 19.2%, 18.0%, and 16.9%) (Figure 1a–d). The application of FA was more effective on the drought-sensitive varieties. In addition, the aboveground dry matter and yield of Yanke 2 were significantly higher than those of Bayou 9 under the DS and DF treatments (Figure 1a–d).

3.2. Effect of FA on Antioxidant Enzyme Activity in Oat Leaves Under Drought Stress

Variety, moisture treatment, and their interactions significantly regulated antioxidant enzyme activities (Table S3). Drought stress (DS) significantly affected antioxidant enzyme activities in the leaves of the two oat varieties, but the response patterns differed among the varieties. GPX, APX, GR, and DHAR activities were significantly reduced by 18–29.75% in Yanke 2 leaves but significantly increased by 29.94–34.98% in the leaves of Bayou 9 under drought stress compared with WW. On the contrary, the GPX, APX, DHAR, and GR activities of Bayou 9 leaves were significantly increased by 29.94–34.98% under drought stress, by 29.97%, 29.94%, 32.24%, and 34.98%, respectively, compared with WW (Figure 2a–d). FA significantly alleviated the effects of drought stress on the antioxidant enzyme activity in oat leaves (p < 0.05). Compared with plants sprayed with fresh water (DS) under drought stress, the activities of GPX, APX, DHAR, and GR in the leaves of Yanke 2 increased by 28.09%, 24.17%, 19.19%, and 26.48%, respectively, and those of Bayou 9 increased by 35.01%, 12.41%, 22.10%, and 33.29%, respectively, when sprayed with FA (DF) (Figure 2a–d). The effect was more significant for the drought-sensitive varieties. Under WW treatment, the GPX, APX, and GR activities of Yanke 2 were significantly higher than those of Bayou 9, whereas the GR and DHAR activities of Bayou 9 were significantly higher than those of Yanke 2 under both DS and DF treatments (Figure 2a–d).

3.3. Effect of FA on Non-Enzymatic Antioxidants in Oat Leaves Under Drought Stress

Variety, moisture treatments, and their interactions significantly regulated the non-enzymatic antioxidant content; moisture treatments and variety–moisture treatment interactions significantly affected AsA/DHA and GSH/GSSG ratios; and variety had a significant effect only on AsA/DHA (Table S3). Drought stress (DS) resulted in a significant increase in DHA content in the leaves of Yanke 2 and Bayou 9, along with a significant decrease in AsA/DHA, compared with WW (Figure 3b,c). Among them, the DHA contents of Yanke 2 and Bayou 9 increased by 15.77% and 52.91%, respectively, and AsA/DHA decreased by 31.66% and 23.75%. In addition, the AsA content of Yanke 2 was significantly decreased under drought stress compared with the normal water supply, whereas the AsA content of Bayou9 was significantly elevated (Figure 3a). Spraying FA under drought stress (DF) significantly increased AsA content and AsA/DHA in the leaves of Yanke 2 and Bayou 9, while DHA content was significantly reduced, with the magnitude of change being greater in Bayou 9 than in Yanke 2 (Figure 3a–c). Drought stress (DS) significantly affected glutathione metabolism in the leaves of the two oat varieties, but the response patterns differed among varieties (Figure 3d–f). The GSH content and GSH/GSSG ratio showed a significant decrease in Yanke 2 leaves and a significant increase in Bayou 9, and GSSG content showed a significant increase in Yanke 2 leaves and a significant decrease in Bayou 9. The FA foliar spray significantly improved glutathione metabolism in both varieties under drought stress (Figure 3d–f). Compared with DS, spraying FA (DF) under drought stress significantly increased the GSH content and GSH/GSSG ratio of Yanke 2 leaves by 26.69% and 37.72%, respectively, while the GSSG content was reduced by 8.36% but the difference was not significant. The GSH content, GSH/GSSG ratio, and GSSG content of leaves of Bayou 9 were significantly increased by 31.68%, 15.30%, and 14.32%. The results showed that FA treatment could effectively alleviate the negative effects of drought stress on glutathione metabolism, increase the GSH content and the GSH/GSSG ratio, and have a greater effect on the drought-sensitive variety. Under WW treatment, the GSH content in the leaves of Yanke 2 was significantly lower than that of Bayou 9, whereas the AsA/DHA and GSH/GSSG ratios were significantly higher than those of Bayou 9. There were significant differences in antioxidant content and the GSH/GSSG ratio between the two varieties under DS and DF treatments, in which only the content of GSSG was significantly higher in the leaves of Yanke 2 than that of Bayou 9, and the content of AsA, DHA, GSH, and the GSH/GSSG ratio were lower than those of Bayou 9 (Figure 3a–d).

3.4. Effect of FA on Chlorophyll Content and Its Intermediates in Oat Leaves Under Drought Stress

Variety, moisture treatments, and their interactions all significantly regulated chlorophyll content (Table S3). Compared with WW, drought stress (DS) resulted in a significant decrease in chlorophyll content in both oat varieties. And the decrease of Yanke 2 was significantly lower than that of Bayou 9. The chlorophyll content of both oat varieties was significantly reduced under drought stress compared to normal water supply conditions (WW). The decrease of Yanke 2 was significantly lower than that of Bayou 9 (Figure 4a–c). This indicated that drought stress impeded chlorophyll synthesis in oat leaves and had a greater effect on Bayou 9. However, spraying FA significantly alleviated the negative effect of drought stress on chlorophyll content. Spraying with FA (DF) significantly increased Chl a, Chl b, and total chlorophyll content in both varieties, and recovered 12.40%, 14.58%, and 12.81% loss in Yanke 2 and 15.13%, 13.88%, and 14.87% loss in Bayou 9, respectively, which was more effective in drought-sensitive varieties. Chl b and total chlorophyll contents of Bayou 9 were significantly higher than those of Yanke 2 under WW, DS, and DF treatments, while Chl a content was significantly higher than that of Yanke 2 only under WW and DS treatments (Figure 4a–c).

Variety significantly regulated the Mg-Proto content, and variety, moisture treatments, and their interactions significantly affected the Mg-Proto, Proto IX, and Pchlide content (Table S3). Compared with WW, drought stress (DS) significantly reduced the levels of Mg-Proto, Proto IX, and Pchlide in the two varieties of oats, with Yanke 2 showing a lesser reduction than Bayou 9 (Figure 4d–f). Under drought stress, spraying FA (DF) significantly increased the levels of Mg-Proto, Proto IX, and Pchlide in the leaves of the two oat varieties; Yanke 2 restored 9.73%, 10.38%, and 12.87% of the losses, respectively; Bayou 9 recovered 16.91%, 11.45%, and 17.13% of the losses, respectively; and drought-sensitive varieties recovered more than drought-resistant varieties (Figure 4d–f). In addition, only Bayou 9 had a significantly higher Mg-Proto content than Yanke 2 under WW and DF treatments (Figure 4d–f).

3.5. Effects of FA on Carbon-Assimilating Enzymes in Oat Leaves Under Drought Stress

Moisture treatments significantly regulated Rubisco, RCA, SBPase, FBA, and TK activities, and variety had a significant effect on RCA, FBA, and TK activities (Table S3). Compared with WW, drought stress (DS) significantly reduced Rubisco, RCA, SBPase, FBA, and TK activities in the two oat leaf varieties, with Yanke 2 showing a smaller reduction than that of Bayou 9 (Figure 5a–e). Under drought stress, spraying FA (DF) significantly increased Rubisco, RCA, FBA, and SBPase in the leaves of the two oat varieties and had no significant effect on TK activity (Figure 5a–e), in which Yanke 2 restored 16.28%, 16.12%, 16.22%, and 18.86% of the losses, and Bayou 9 recovered 16.96%, 17.72%, 17.81%, and 18.99% of the losses, and FA had a stronger mitigating effect on drought-sensitive varieties. Under DF treatment, only FBA activity differed significantly between the two varieties; under DS treatment, RCA and SBPase differed significantly between the two varieties; under DF treatment, only TK activity differed significantly between the two varieties; and the values of the above indexes were higher in Yanke 2 than in Bayou 9 (Figure 5a–e).

3.6. Effect of FA on Carbon Metabolism in Oat Leaves Under Drought Stress

Variety significantly affected SPS, SS, and NI activities, and moisture treatments significantly affected SPS, SS, S-AI, and NI activities, whereas variety-water treatment interactions only affected NI activities (Table S3). Compared with WW, drought stress (DS) significantly reduced SPS and SS activities and significantly increased S-AI and NI activities in the leaves of both oat varieties, with the larger change in Bayou 9 (Figure 6a–d). Under drought stress, FA (DF) significantly increased the SPS, SS, and AI activities in the leaves of two varieties of oats and the NI activity in the leaves of Bayou 9, whereas the effect of NI activity in the leaves of Yanke 2 was insignificant (Figure 6a–d), with the increases in the SPS, SS, and AI activities being 16.50%, 18.06%, and 13.50% in the leaves of Yanke 2 and 22.09%, 23.64%, 19.75%, and 14.65% in the leaves of Bayou 9. It was indicated that FA increased the activities of carbon metabolism-related enzymes in oats under drought conditions. Under DS treatment, the SPS and SS activities of Yanke 2 were significantly higher than those of Bayou 9, while the NI was significantly lower than that of Bayou 9. Under DF treatment, the SS activity of Yanke 2 was significantly higher than that of Bayou 9, while the NI was significantly lower than that of Bayou 9 (Figure 6a–d).

Moisture treatment significantly affected glucose, sucrose, and fructose content and variety significantly affected glucose and sucrose content (Table S3). Drought stress (DS) significantly reduced the sucrose content and significantly increased the glucose and fructose content (p < 0.05) in both oat varieties (Figure 7a–c). Compared with WW, the sucrose content in the leaves of Yanke 2 and Bayou 9 plants decreased by 26.19% and 27.89%, while the glucose content increased by 26.84% and 28.89%, and the fructose content increased by 22.46% and 23.17%. Under drought stress, FA (DF) significantly increased the sucrose, glucose, and fructose contents in the leaves of two oat varieties (Figure 7a–c), and the increases in the three sugars in the leaves of Yanke 2 and Bayou 9 were 18.16%, 17.96%, and 22.41%, and 22.05%, 20.67%, and 25.11%, when compared with that of sprayed fresh water (DS). The sucrose content of Yanke 2 was significantly higher than that of Bayou 9 under WW and DS treatments (Figure 7a).

3.7. Correlation Analysis of Growth and Physiological Indexes Under Different Treatments

Correlation analysis showed (Figure 8a,b) that the growth and yield indices of both oat varieties showed positive correlations with chlorophyll and its intermediates and also strongly correlated with GSH/GSSG, GSSG, and DHA, with Yanke 2 showing a positive correlation and a significant negative correlation with GSSG and DHA (p < 0.05), while the opposite was true for Bayou 9. Aboveground dry matter and grain yield were correlated with APX, with a positive correlation in Yanke 2 and a negative correlation in Bayou 9 (p < 0.05), indicating that the different genotypic varieties responded differently to drought stress and FA. As shown in Figure 8b,c, the growth and yield indices of the two varieties of oats were positively correlated with the five carbon-assimilating enzymes (Rubisco, RCA, SBPase, FBA, and TK), as well as SPS, SS, and sucrose, and were weakly correlated with S-AI, fructose, and glucose, while the growth and yield indices of aboveground dry matter and grain yield were negatively correlated with NI (p < 0.05).

3.8. Hierarchical Cluster Analysis of Growth and Physiological Indicators Under Different Treatments

Hierarchical cluster analysis showed (Figure 9) that drought stress (DS) inhibited the growth and yield of both varieties of oats and reduced chlorophyll and its intermediates’ content, carbon–assimilating enzymes (Rubisco, RCA, SBPase, FBA, TK), SPS, SS, and sucrose, as well as AsA/DHA, whereas S-AI, NI activity, fructose and glucose content, and DHA were significantly higher than in the normal water supply (WW). In addition, GPX, APX, GSH, DHAR, GR, AsA, and GSH/GSSG in the leaves of Yanke 2 showed a decreasing trend, and DHA and GSSG showed an increasing trend under drought stress, whereas the performance of Bayou 9 was in the opposite direction. Under drought stress, FA application significantly increased the plant growth and yield of both oat varieties and also enhanced carbon assimilative, carbon metabolizing, and antioxidant enzyme activities; increased chlorophyll and its intermediates, carbohydrates, and antioxidant content (AsA, GSH), while DHA was decreased. In contrast, GSSG decreased in Yanke 2 leaves and increased in Bayou 9 leaves in response to FA. This suggests that under drought stress, FA promotes oat growth and increases yield by regulating the antioxidant system and carbohydrate metabolism, promoting chlorophyll synthesis, and improving carbon–assimilation capacity. However, the two varieties responded to FA in different ways, with the drought-sensitive variety being more sensitive to FA.

4. Discussion

4.1. Effect of FA Treatment on Growth and Yield Under Drought Stress

Studies have shown that FA effectively mitigates the inhibitory effects of abiotic stresses on plant growth [39,40]. In this experiment, we found that drought stress significantly reduced leaf area, leaf dry matter, aboveground dry matter accumulation in oat leaves, and yield. In contrast, the aboveground dry matter mass and yield of both oat varieties were significantly increased after spraying with FA under drought stress, which is consistent with the findings of Ibtisam et al. [39] in barley. The mechanism of action may be due to the small molecular weight of FA, which can penetrate the cell membrane and form complexes with cations to promote nutrient uptake, thereby enhancing dry matter accumulation and increasing yield [41,42]. In addition, FA significantly enhanced leaf area and leaf dry matter only in the drought-sensitive variety, whereas it had no significant effect on the drought-tolerant variety. This suggests that FA may preferentially activate drought-response mechanisms in the sensitive variety, a phenomenon similar to the findings of melatonin regulation of drought tolerance in which melatonin significantly increased drought tolerance in sensitive peanuts but had no significant effect on the phenotype of the drought-tolerant variety [43].

4.2. Effect of FA on AsA-GSH Cycling in Oat Leaves Under Drought Stress

Differential responses of the two oat varieties to drought stress were observed in this experiment: leaf AsA content was reduced in Yanke 2, whereas it was elevated in Bayou 9. This difference may stem from the fact that AsA was oxidized to MDHA and DHA by MDHAR and DHAR in Yanke 2 when ROS were produced in large quantities [44], and at the same time, its DHAR activity was reduced, resulting in blocked AsA regeneration, which explains why DHA was elevated under drought stress; Bayou 9, on the other hand, improved AsA regeneration efficiency due to enhanced DHAR activity. GSH participates in ROS scavenging through the AsA-GSH cycle and is oxidized to GSSG after scavenging H₂O₂ [44]. In this study, Yanke 2 exhibited decreased GPX and GR activities and GSH content and increased GSSG content under drought stress, whereas Bayou 9 showed the opposite trend. This difference may arise from the regulation of the reduction of GSSG to GSH by GR [45]. Studies have shown that humic acid analogs activate protective enzymes such as APX, GR, and GPX and promote AsA and GSH synthesis [46]. In this experiment, FA treatment increased the AsA and GSH content and APX, GR, and DHAR activities in oat leaves under drought stress while reducing the DHA content. The changes in GR activity and GSH content were consistent with the pattern of change, indicating that the high activity of GR is a guarantee for the increase of GSH. FA maintains the efficient operation of the AsA-GSH cycle by enhancing the activity of antioxidant enzymes and promoting the regeneration of antioxidants so as to achieve a balance between the production and removal of ROS and to increase the ability of antioxidant stress to alleviate the inhibition of the growth of the plant by drought stress, which is consistent with the findings of Shen Jie et al. [47] in millet.

The AsA/DHA and GSH/GSSG ratios are important indicators of the redox state of plant cells and can dynamically characterize changes in the redox state of AsA and GSH under adversity stress [48], which is particularly important for maintaining cellular redox homeostasis under abiotic stress [49]. In this study, the AsA/DHA and GSH/GSSG ratios in the leaves of the two oat varieties decreased significantly under drought stress, whereas the GSH/GSSG ratio increased significantly in the leaves of Bayou 9. This suggests differences in tolerance strategies to redox imbalance across genotypes. Spraying FA increased AsA/DHA and GSH/GSSG, indicating that spraying FA could increase the ratios of AsA/DHA and GSH/GSSG in oat leaves to reduce the oxidative damage caused by drought. This result reveals that FA promotes the conversion of oxidative AsA (DHA) and GSSG to the reduced state by regulating the activities of key enzymes of the AsA-GSH cycle (e.g., APX, GR), thereby enhancing the antioxidant buffering capacity of cells [50]. The enhancement of AsA-GSH cycling activity resulted in a stronger ROS scavenging ability and protection system, inhibited the degree of membrane lipid peroxidation, mitigated the damage caused by drought to the cell membrane, and re-established the dynamic balance of intracellular ROS, which ultimately improved the photosynthetic function of the leaves [51].

4.3. Effect of FA on Chlorophyll Synthesis in Oat Leaves Under Drought Stress

Drought not only reduced chlorophyll content but also inhibited the accumulation of some key biosynthetic precursor molecules in porphyrin or chlorophyll metabolism, including 5-aminolevulinic acid, cholorophyllogen, Proto IX, Mg-Proto IX, and Pchlide [52]. The present study showed that the chlorophyll a, b, and total chlorophyll content of leaves of both oat varieties decreased under drought stress, and the decrease was more pronounced in Bayou 9, which is similar to the results of Dalal et al. [53] in rice that a water deficit leads to the blockage of chlorophyll biosynthesis by inhibiting the accumulation of key precursors of the porphyrin metabolic pathway. Moreover, drought stress significantly reduced the magnesium Mg-Proto, Proto IX, and Pchlide contents in both varieties, indicating that the drought caused the chlorophyll synthesis of oat leaves to be blocked, resulting in the weakening of photosynthetic capacity and the inhibition of plant growth [43]. The reason for this may be that under drought stress, the leaves of the plant wilted, and the structure of the vesicle membrane was damaged, which hindered chlorophyll biosynthesis and decreased the content of photosynthetic pigments, and further indicated that Bayou 9 was more sensitive to drought stress [54]. Previous studies have shown that spraying FA increases the photosynthetic pigment content of lettuce under adversity stress [29]. In this experiment, we found that spraying FA under drought stress significantly increased the content of chlorophyll a, chlorophyll b, and total chlorophyll in oats, and the levels of the intermediate products of chlorophyll synthesis, Mg-Proto, Proto IX, and Pchlide, were also significantly increased, suggesting that FA enhances the synthesis of chlorophylls under drought stress by regulating the chlorophyll precursors. The reason for this may be that FA maintains normal chloroplast ultrastructure and promotes chlorophyll synthesis [32].

4.4. Effect of FA on Carbon-Assimilating Enzymes in Oat Leaves Under Drought Stress

Water-deficit stress impairs photosynthetic carbon assimilation primarily by disrupting the activity of key enzymes in the Calvin cycle. As reported previously [55], drought-induced declines in Rubisco and RCA activities directly compromise leaf gas exchange and photosynthetic efficiency. Consistent with this, our study observed Rubisco and RCA were significantly decreased under drought stress, which may be attributed to the decrease in the amount of the corresponding enzymes due to drought stress, which led to a decrease in Rubisco activity and a decrease in photosynthetic carbon assimilation [56]. It was shown that FA significantly affected the activities of Rubisco, RCA, the Rubisco large subunit, the small subunit, and RCA gene expression in cucumber leaves under drought stress [32]. Jannin et al. [57] suggested that the increase in Rubisco activity after humic acid treatment may be caused by an increase in the number of chloroplasts in the cells. In this study, we found that under drought stress, FA significantly increased the enzyme activities of Rubisco and RCA in oat leaves, and the enhancement effect was more significant, especially for the drought-sensitive variety. The mechanism of its action may be that FA mitigates the oxidative damage of drought on chloroplasts by maintaining the integrity of the membrane structure of the quasi-vesicles and provides a stable microenvironment of enzyme activities for Rubisco and RCA. The results were similar to those of Doron L et al. [58] in maize, during to which the increase in Rubisco content significantly improved the photosynthetic capacity of maize and enhanced its drought tolerance. SBPase is involved in the reductive pentose phosphate cycle during photosynthesis and controls carbon influx and regeneration during the Calvin cycle. A small decrease in SBPase activity can inhibit CO₂ fixation [59]. In this experiment, the SBPase activity of oat leaves of both varieties was reduced under drought stress, whereas FA alleviated the reduction in SBPase activity, suggesting that FA can maintain the efficiency and ability of oat plants in fixing CO₂ under drought stress and promote the glycolytic metabolism to proceed smoothly. FBA and TK, non-regulated enzymes that catalyze reversible reactions in the Calvin cycle, indirectly affect the efficiency of CO₂ fixation by controlling the flux of C₃ cycle intermediates [60]. In this study, we found that the FBA and TK activities of the leaves of two oat varieties decreased significantly under drought stress. FA inhibited the decrease in FBA activity in oat leaves under drought stress and had less effect on TK activity, which was better for the drought-sensitive variety, suggesting that FA regulates the activities of key enzymes of carbon assimilation to maintain the smooth progress of the photosynthetic carbon-assimilation process in oats under drought stress.

4.5. Effect of FA on Carbon Metabolism-Related Enzymes in Oat Leaves Under Drought Stress

Drought stress can alter the partitioning of photosynthetically fixed carbon between starch and sucrose by affecting SPS and SS activity [61]. Some sucrose molecules are broken down by NI and S-AI to produce glucose and fructose to maintain normal leaf metabolism. The results of the present study showed that drought stress significantly reduced the SPS and SS activities of Yanke 2 and Bayou 9 but increased the S-AI and NI activities. This indicated that the sucrose synthesis capacity is lower than the catabolic capacity, which reduces sucrose content and contributes to the breakdown of sucrose into glucose and fructose, leading to the accumulation of glucose and fructose in the leaves, similar to the findings of Du et al. [62] in soybean. Whereas, it was also shown that drought significantly elevated soybean SPS and SS [63], contrary to the results of the present experiment, and the difference might be related to the different drought intensity, duration, stress period, and crop type. Compared with Bayou 9, the decrease in SPS and SS and the increase in NI and S-AI activities in the leaves of Yanke 2 under drought stress were smaller, which was favorable to the accumulation of sucrose. This indicates that the drought resistance of oats is closely related to the content of non-structural carbohydrates. In this study, we found that FA spraying increased SPS, SS, S-AI, and NI activities in the leaves of two oat varieties under drought stress, which indicated that FA regulates the synthesis and decomposition rate of sucrose through bidirectional regulation of the key enzyme activities of sucrose metabolism and optimizes the homeostatic distribution of oat sugars, thus enhancing its drought resistance.

4.6. Effect of FA on Carbohydrate Content in Oat Leaves Under Drought Stress

Changes in the soluble sugar content of crop leaves are determined by the glucose and fructose contents, and high concentrations of hexose not only keep cell expansion pressure stable but also inhibit the feedback of photosynthesis [64]. The results of this study showed that drought stress significantly reduced the sucrose content and significantly increased the glucose and fructose contents in the leaves of the two oat varieties, indicating that drought stress promotes the production of total soluble sugars in oat leaves, which balances the osmotic potential and water balance of the cells in the plant and contributes to the maintenance of normal physiological activities in oats under drought stress [65]. The glucose and fructose contents of Bayou 9 increased more than those of Yanke 2 under drought stress, indicating that the osmoregulatory effect of soluble sugars and its feedback effect on photosynthesis were more obvious in Bayou 9 under drought stress. Spraying FA could promote sucrose synthesis and reduce fructose and glucose content in oat leaves under drought stress, indicating that FA could enhance metabolic capacity and protect structural stability by regulating the synthesis and decomposition of carbohydrates in oat leaves under drought stress, which in turn promotes photosynthetic phosphorylation, strengthens the capacity of chloroplasts to assimilate photosynthetic carbon, and maintains photosynthetic activity.

4.7. The Agronomic Implications of FA

FA induces partial closure of crop stomata and reduces water loss from the stomata, thereby prolonging water utilization by the crop [33], a mechanism that provides direct relief from crop water stress in drought-prone marginal areas where irrigation is restricted, allowing the crop to maintain physiological functions under intermittent drought or limited water supply conditions. It has been shown that FA can significantly increase the N, P, and K content of pea tissues, and the crop can absorb nutrients directly from the leaves, which improves nutrient utilization and reduces the need for heavy soil fertilizer application, which is particularly important in arid marginal areas [66]. The foliar uptake pathway of FA can circumvent the problem of low fertilizer effectiveness due to water scarcity in the soils and environmental pollution due to over-fertilization, which can help to achieve crop nutrient requirements with lower inorganic fertilizer inputs in this type of area, reducing production costs and the risk of face source pollution. FA, as a natural biostimulant, reduces drought damage to cells by inducing antistress substances such as proline and malondialdehyde and antioxidant enzyme systems such as superoxide dismutase [33,67]. In arid and marginal areas, crops often face frequent short-term drought stress, and traditional antiretroviral measures (e.g., large-scale irrigation, soil conditioners) are costly and difficult to implement, whereas the foliar application of FA, which is easy to operate and quick to produce results, can rapidly enhance the crop’s own resilience, making it an effective supplement to traditional measures.

5. Conclusions

Drought stress inhibits oat growth and development and hinders dry matter accumulation, leading to reduced yield. Under drought stress conditions, FA promotes growth and dry matter accumulation and increases oat yield by regulating the ascorbate–glutathione cycle, promoting chlorophyll synthesis, increasing carbon-assimilative enzyme activity, and enhancing carbohydrate metabolism. FA is more effective on the water-sensitive Bayou 9. This study revealed several aspects of the favorable effects of FA treatment on oat growth.