Combined action of salicylic acid and silicon regulates osmoregulatory substances and antioxidant enzymes to improve drought tolerance and yield in dry-cultivated rice

doi:10.21203/rs.3.rs-4948148/v1

Salicylic acid (SA) and silicon (Si) protect against a variety of stresses. Our group found that Si improved the drought tolerance of dry-cultivated rice. No studies are available regarding the synergistic effect of aboveground SA spraying and belowground Si application on the drought tolerance and yield of dry-cultivated rice, necessitating further research. Two rice types with different levels of drought resistance (Suijing18 and Hongke88) were selected for this study. The optimal SA concentration (1 mM/L) was determined via five different SA spraying treatments at various concentrations. Then SA and Si were combined, which included a Control, 45 kg/hm² Si, 1 mM SA, and 1 mM SA + 45 kg/hm² Si to examine their synergistic effect on the morphological characteristics, yield, osmotic regulatory substances, chlorophyll fluorescence, and antioxidant enzymes of two different drought-resistant rice cultivars. The results showed that combined SA + Si application increased the plant height, root length, aboveground dry weight, and belowground dry weight of these two drought-resistant rice types. The yield of which increased by 80.70% and 77.26%, respectively. Compared with the control, while the photosynthetic capacity improved significantly, the minimum photochemical efficiency (Fv/Fm) values increased by 30.97% and 19.17%, while the photochemical quantum yield (ΦPSII) values were 29.01% and 29.64% higher, the NPQ values decreased by 67.55% and 39.98%, respectively. Moreover, treatment increased the soluble sugar and soluble protein levels, osmoregulatory capacity, antioxidant enzyme activity, and increased endogenous SA + Si content in the two dry-cultivated rice species. Hence, the combined application of SA and Si improves the drought resistance of dry-cultivated rice by increasing the content of osmoregulatory substances, enhancing the levels of endogenous SA and Si, upregulating the activity of antioxidant enzymes, and strengthening the photosynthetic capacity of the PSII system. This results in improved drought-resistant capability and increase of the accumulation of dry matter and yield of dry-cultivated rice, which produces a more significant alleviation of the adverse effects of drought stress compared to the application of either substance alone.

Biological sciences/Plant sciences

Biological sciences/Plant sciences/Plant breeding

Biological sciences/Plant sciences/Plant hormones

Biological sciences/Plant sciences/Plant physiology

Biological sciences/Plant sciences/Plant stress responses

Dry-cultivated rice

Salicylic acid

Silicon

Osmotic regulator

Antioxidant oxidase

Chlorophyll fluorescence

The global rise in the incidence and severity of drought significantly constrains food production. It is critical to prioritize the development of drought-resistant crops to mitigate the negative impact of drought on agricultural yields as global populations increase and water resources are depleted.¹ Dry-cultivated rice has the potential to address the issues of labor and water resource shortages while ensuring the sustainable cultivation of rice. As an efficient, resource-saving, mechanized, climate-smart, and economically viable strategy², it is becoming an alternative to irrigated rice cultivation.³ However, the drought tolerance of dry rice must be improved to increase yields.

Plants are especially susceptible to drought during the reproductive stage. In cereal crops, particularly rice, this stage is highly sensitive to water restrictions.⁴ The reduced rice grain yield during this stage in drought conditions is associated with changes in the metabolism and redox state of its apical leaves.⁵ The drought-induced decrease in carbon assimilation enhances the production of reactive oxygen species (ROS) in leaf cells.⁶ Excess ROS accumulation can decrease grain yield and leaf senescence due to photosynthetic pigment degradation.⁷ The typical positive correlation between photosynthesis and crop production significantly affects the growth and development of rice and reduces yield.⁸ Salicylic acid (SA) accumulation was found to play a role in the defense signaling and regulation of cell death.⁹ SA is an endogenous small-molecule phenolic compound that acts as a signaling sensor to regulate plant responses. It protects plant cells from ion accumulation toxicity and cell death by managing processes such as antioxidant defense, nitrogen metabolism, photosynthesis, and water stress.¹⁰ Pretreating rice roots with SA promotes root growth, reduces ROS levels and membrane damage, and increases superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activity, as well as glutathione (GSH) and chlorophyll content, while reducing MDA levels.¹¹ Exogenous SA application alleviates oxidative stress in drought-stressed plants by modulating important enzymatic and non-enzymatic pathways.¹² In addition, the involvement of SA in glycolytic/TCA cycling, starch and sucrose metabolism, and antioxidant pathways reverses the rice yield and grain weight caused by soil drought.¹³ SA can also increase the photosynthetic capacity of plants.¹⁴ Studies have shown that exogenous SA application increases the rate of photosynthesis in sweet potatoes and reduces drought damage.¹⁵ Exogenous SA spraying also significantly elevates the PSI and ETR levels in T. ciliata, which increases the electron transfer and recovery rates of PSII reaction center activity, ultimately enhancing photosynthesis.¹⁶

Silicon (Si), the second most important element in soil, typically accumulates in rice at significantly higher levels than nutrients such as nitrogen, phosphorus, and potassium.¹⁷ Si improves the resistance of plants to various abiotic stresses by regulating the synthesis and accumulation of endogenous hormones. During abiotic stress-induced adversity, Si helps to control multiple plant metabolic processes, including osmotic stress generation and ROS regulation, via the antioxidant defense system.¹⁸ Si also promotes plant growth by increasing the photosynthetic rate and nutrient uptake, improving plant defense responses, and enhancing tolerance to a range of stresses.¹⁹ Furthermore, Si can increase rice spike formation and grouting rates to enhance rice yields.²⁰

Since the large consumption of freshwater resources constrains sustainable agricultural development, dry-cultivated rice provides a viable alternative to reduce water utilization during cultivation. Therefore, current research focuses on improving dry-cultivated rice yields. Although both SA and Si can protect plants from biotic and abiotic stresses, minimal studies are available regarding the effect of applying these substances simultaneously to improve the drought tolerance and yield of dry-cultivated rice. Our research team has previously demonstrated that moderate amounts of Si fertilizer can improve the yield and quality of dry-cultivated rice by regulating source supply capacity and seed starch synthesis.²¹ The present study focuses on the impact of combined exogenous SA and Si application on the morphological characteristics, yield, chlorophyll fluorescence, antioxidant enzyme activity, and Si and SA content in dry-cultivated rice.

2.1. Plant materials and treatments

This study used two high-quality dry-cultivated rice varieties from Jilin Province, namely China-Suijing18 with strong drought resistance and Hongke88 with weak drought resistance. A pot experiment was conducted in 2022–2023 at Jilin Green and High Quality Japonica Rice Engineering Research Center. The soil contained a moderate level of Si at 113.46 mg kg^− 1, an organic carbon content of 9.60 g kg^− 1, an alkali-hydrolyzed nitrogen content 33.89 mg kg ^− 1, an available potassium content of 137.09 mg kg ^− 1, an available phosphorus content of 29.42 mg kg ^− 1, and a pH value of 6.70.

An experiment was conducted in 2022 using small three-holed basins of 14 cm high and 15 cm in diameter, with each hole containing five rice grains. Different SA concentrations (0 mM/L, 0.01 mM/L, 0.5 mM/L, 1 mM/L, 1.5 mM/L, and 2 mM/L) were sprayed onto the leaf surfaces of the rice seedlings during the three-leaf single-phase stage. Measurements were recorded at 3h, 6h, 9h, 12h, 24h, 36h, and 72h. The leaves were only sprayed until moist but not dripping. Then, the changes in the samples were monitored and recorded to determine the optimal SA concentration. The experiment used a fully randomized design with five replicates.

In 2023, large basins with heights of 22 cm and diameters of 30 cm were used for rice cultivation. Each basin had three holes, with each hole containing eight rice grains. A random block design was adopted, and each treatment was repeated five times while the growth period was mainly rainfed. The experiment used 75 kg/hm² phosphate fertilizer (calcium superphosphate, P₂O₅ 12%) (equivalent to P₂O₅) and potassium fertilizer (potassium chloride, K₂O 60%) (equivalent to K₂O), as well as 160 kg/hm² nitrogen fertilizer (urea, pure N 46%) (equivalent to pure N). Biolli Russian mineral Si fertilizer was added at an effective level of ≥ 72% and an application amount of 45 kg/hm², while the optimal SA concentration determined in the 2022 experiment was sprayed during the booting stage. Diseases, pests, and grasses were strictly controlled to prevent production loss. The two dry-cultivated rice varieties were divided into four treatment groups: control group, 45 kg/hm² Si, 1 mM SA, and 1 mM SA + 45 kg/hm² Si. The Suijing18 strain was represented by S1, S2, S3, and S4, while the Hongke88 cultivar was represented by P1, P2, P3, and P4. The experiment used a fully randomized design with five replicates.

2.2 Assessment of the growth and yield parameters

Five plants were sampled from each plot at the maturity stage to determine dry matter accumulation. The samples were deoxidized in an oven at 105℃ for 30 min and dried to a constant weight at 80℃, after which the samples were weighed. The dry matter weight was determined using the 1% balance method. The root length measurements were repeated three times for each treatment. Three pots of rice were carefully washed with tap water and then rinsed with pure water. The roots were straightened, and the distance from the root base to the tip was measured using a ruler. The plant height was determined using a tape measure. The distance from the base of the rice plant to the top leaf was measured and averaged. The effective panicle number of each point was measured in each pot at the maturity stage. Five plants with the same average panicle number were selected for seed testing after drying. Furthermore, the total number of grains per spike, the number of solid grains, the 1000-grain weight, and the seed setting rate were determined. After harvesting and drying, the rice quality and water content were measured, and the rice yield was calculated according to a standard water content of 13.5%.

2.3 Chlorophyll fluorescence assay

Three plants showing uniform flag leaf growth were selected for each treatment. Following a 24-h spray application of SA treatment, a portable chlorophyll fluorescence analyzer (PAM-2500, Shanghai, China) was used to determine the fluorescence parameters of each labeled pot leaf in the dark from 20:00 to 23:00 on the night before the measurement date. The fluorescence parameters were determined in natural light from 9:00 to 12:00 the next day. The fluorescence indexes included the maximum photochemical efficiency (Fv/Fm), the photochemical quantum yield (ΦPSII), the non-photochemical quenching coefficient (NPQ), and the photochemical quenching coefficient (qP).

2.4 Determination of the soluble sugar and soluble protein content

Si, SA, and SA + Si were sprayed during the booting stage, and leaf samples were collected for testing at 2 h, 24 h, and 10d, respectively. Next, 0.1 g of the crushed, sifted dried plant samples were weighed into a 2 mL EP tube, after which 80 µL of 80% ethanol was added. The samples were sealed and boiled in a water bath at 80℃ for 30 min. The soluble sugar content of the collected materials was determined using the anthranone sulfate colorimetric method.

Next, after Si, SA, and SA + Si spray treatment during the booting stage and leaf-sample testing at 2 h, 24 h, and 10d, respectively, 0.1 g was weighed, frozen in liquid nitrogen, ground into a powder, and thoroughly mixed after adding 1 mL of TBS. The mixture was centrifuged at 14,000 rpm and 4℃ for 5 min, after which the supernatant was collected as the total soluble protein extract. A Bradford protein assay (Bio-Rad) was used to determine the protein concentration, with bovine serum albumin as the standard. The total soluble protein content in one sample was calculated according to the protein concentration and protein extract volume. The average value of the three seedlings in each experiment was determined.²²

2.5 Determination of the antioxidant enzyme activity

All the chemical and enzyme reagents used to determine the grain enzyme activity were obtained from Solarbio Chemical Company. Here, 5 mL of 50 mM HEPES buffer (PH7.8, 20% (v/v) glycerol, 1 mM EDTA, 1 mM ASA, 1 mm GSH, 5 mM MgCl₂, and 1mM DTT were added to a 0.5 g of the leaves. The sample was ground, extracted in an ice bath, homogenized at 4℃, and centrifuged at 10,000 g for 20 min, after which the supernatant was collected for analysis to determine the activity of the various enzymes. The SOD activity was determined using a reaction system consisting of 1.5 mL of 0.05 M pH 7.8 HEPES buffer, 0.3 mL of 130 mM methionine, 0.3 mL of 750 mM nitro blue tetrazole, 0.3 mL of 100 µM EDTA, and 0.3 mL of 60 µM riboflavin. Buffer was used as a blank control instead of an enzyme solution. After irradiation at 25℃ and 4,000 lx for 15 min, the blank color changed from yellow to blue. The reaction was terminated immediately in dark conditions, after which the OD₅₆₀ absorption value was determined immediately. The mixture for determining the POD consisted of 1 mL of sodium phosphate buffer (pH 7.8), 0.95 mL of 0.2% guaiacol, 1 mL of 0.3% H₂O_2, and 0.05 mL of the enzyme extract. The absorbance was determined at 470 nm for 90 s with an interval of 30 s. One unit of the POD activity was defined as the amount of enzyme that caused the decomposition of 1 mg of the substrate at 470 nm.²³ The ascorbate peroxidase (APX) and glutathione reductase (GR) activity was determined using an ELISA kit (Solarbio, Beijing, China). The CAT activity was measured at 25℃ using a reaction system consisting of 1.9 mL of 50 mM pH 7.0 HEPES phosphate buffer, 1 mL of 22 mM H₂O₂, and 0.1 mL of the enzyme solution. The OD₂₄₀ kinetic change was determined at an interval of 1 min.²⁴

2.6 Determination of the Si and SA content in the dry-cultivated rice

Here, 0.1 g of the plant sample was placed in a heating tube, after which 3 mL nitric acid and 1 mL hydrofluoric acid were added. This was followed by a three-stage heating digestion procedure. Stage 1: The temperature was increased to 120℃ for 5 min and stabilized for 5 min. Stage 2: The temperature was increased to 160℃ for 5 min and stabilized for 5 min. Stage 3: Temperature was increased at 180℃ for 5 min and stabilized for 25 min. The heating tube was placed in a graphite acid drive meter, and the temperature was set at 160℃ for the acid drive. Once the heating tube started emitting yellow smoke followed by white smoke, the solution was considered fully acidified when it reached 1–2 mL and was removed after cooling to room temperature. The acid-treated sample was washed 2–3 times and transferred to a 100 mL volumetric bottle for measurement. The Si content in the solution was determined via ICP (ICP1000II, US), while the SA level was measured using an ELISA kit (Boyan, Nanjing, China).

2.7 Statistical analysis

Microsoft Excel 2021 was used for data sorting and processing, while GraphPad Prism 9.5.1 and the ChiPlot online tool were used for plotting. The IBM SPSS Statistics 26 software was used for statistical analysis, and the significance level of each statistical test was expressed as 0.01 < p ≤ 0.05 = *, 0.001 < p ≤ 0.01 = **, 0.0001p < 0.001 = ** *.

3.1 The effect of different SA concentrations on the growth of the dry-cultivated rice seedlings

SA concentrations of 0 mM/L, 0.01 mM/L, 0.5 mM/L, 1 mM/L, 1.5 mM/L, and 2 mM/L were used for this assessment. After treatment, the SOD activity in the leaves of the two rice seedling cultivars reached the highest level at an SA concentration of 1 mM/L after 24 h. The Suijing18 samples in different treatment groups were compared with the control group. The SOD activity increased by 8.45%, 2.41%, 12.25%, 6.59%, and 8.91%, respectively, while the POD activity was 45.53%, 1.25%, 59.44%, 32.08%, and 17.24% higher. CAT activity rose by 22.03%, 12.04%, 28.94%, 28.51%, and 15.66%, respectively, while the GR activity reached a maximum level at an SA concentration of 1 mM, denoting a 223.29% increase. The APX activity increased by 18.37%, 215.46%, 451.61%, 327.41%, and 240.53%, respectively. These results indicated that the activity of five antioxidant enzymes reached the highest value at an SA concentration of 1 mM (Fig. 1a). Next, the Hongke88 samples in the different treatment groups were compared to the control group. The SOD activity increased by 2.89%, 1.71%, 13.53%, 3.61%, and 9,21%, respectively, while the POD activity was 22.29%, 17.12%, 33.87%, 13.98%, and 21.51% higher. The CAT activity rose by 15.55%, 11.82%, 18.41%, 6.80%, and 15.83%, respectively, while GR activity increased by 221.57%, 193.80%, 518.30%, 163.11%, and 19.86%. The APX activity was 134.77%, 198.30%, 367.11%, 11.94%, and 240.66% higher (Fig. 1c). Finally, the concentration of sprayed SA sprayed was determined as 1 mM for subsequent experiments.

The SA concentration was determined, and the activity of five antioxidant enzymes was measured at 3 h, 6 h, 9 h, 12 h, 24 h, 36 h, and 72 h after spraying. The activity of the five enzymes reached maximum levels after 24 h. The Suijing18 and Hongke88 samples displayed maximum SOD activity levels of 857.14 U/g and 826.51 U/g, respectively, which was significantly higher than in the control group. Furthermore, the two varieties exhibited maximum POD activity levels of 140.48 U/g and 124.05 U/g, maximum CAT activity values of 1.23 U/g and 1.14 U/g, and respective maximum APX levels of 4.56 U/g and 4.95 U/g (Figs. 1b and 1d). Moreover, the SA content in two dry-cultivated rice varieties was determined at different concentrations and times. The results indicated that the concentration was 1.0mM and the time was 24h, the SA content reached maximum values of 2819.73 U/g and 2881.95 U/g in the Suijing18 and Hongke88, respectively, at 24 h and an SA concentration of 1 mM (Figs. 1e and 1f). Therefore, an external SA spraying concentration of 1 mM and a determination time of 24 h were used in the subsequent experiment.

3.2 The Growth and Biomass of Dry-cultivated Rice

The two dry-cultivated rice varieties yielded similar growth index values at different treatments. Compared with the control group, exogenous Si, SA, and SA + Si application increased the plant height, root length, aboveground dry weight, and underground dry weight of the rice samples (Table 1). Compared with the S1 treatment group, the heights of the plant samples in the Suijing18 S2, S3, and S4 treatment groups increased by 15.76%, 14.37%, and 17.76% higher, the root lengths by 43.76%, 28.53%, and 51.86%, the above-ground dry weights by 34.20%, 24.18%, and 40%, and the underground dry weights by 42.52%, 19.02%, and 48.93%, respectively. The S4 group showed the most significant increase (Fig. 2a). Compared with the P1 treatment group, the heights of the plants in the Hongke88 P2, P3, and P4 treatment groups increased by 15.96%, 14.76%, and 20.19%, the root lengths by 51.59%, 37.10%, and 57.70%, the above-ground dry weights by 30.92%, 24.02%, and 39.06%, and the underground dry weights by 41.96%, 20.51%, and 53.38% respectively. The most significant increase was evident in the P4 treatment group (Fig. 2b). The Suijing18 dry-cultivated cultivar exhibited higher drought tolerance than Hongke88. Exogenous SA + Si spraying increased the Hongke88 plant height, root length, and underground dry weight by 2.43%, 5.9%, and 4.45%, respectively, which exceeded the values of the Suijing18 samples. Therefore, compared with individual exogenous Si and SA spraying, combined SA + Si application showed a better effect on the dry-cultivated rice, especially on the cultivar with weak drought resistance.

Table 1

Effects of exogenous salicylic acid and silicon on the growth of drought cultivation rice.
Variety	Treatment	Above ground dry weight(g)	Underground dry weight(g)	Plant height(cm)	Root length(cm)
Suijing18	S1	40.15 ± 1.23d	4.68 ± 0.32d	82.67 ± 3.62c	30.85 ± 2.35c
	S2	53.88 ± 4.32ab	6.67 ± 0.65a	95.70 ± 4.53ab	44.35 ± 3.56ab
	S3	49.86 ± 2.32bc	5.57 ± 0.45bc	94.55 ± 5.23ab	39.65 ± 2.38b
	S4	56.21 ± 2.64a	6.97 ± 0.44a	97.35 ± 2.54a	46.85 ± 2.36a
Hongke88	P1	38.68 ± 3.65d	4.29 ± 0.56d	78.95 ± 4.44d	27.68 ± 5.36c
	P2	50.64 ± 2.68b	6.09 ± 0.14b	91.55 ± 5.64ab	41.96 ± 4.23ab
	P3	47.97 ± 2.68c	5.17 ± 0.23c	90.60 ± 4.12b	37.95 ± 4.34b
	P4	53.79 ± 1.68ab	6.58 ± 0.54a	94.89 ± 4.45ab	43.65 ± 1.25ab

Mean values followed by different letters in the same column are significantly different from each other (T- test, p < 0.05). The data represent the means of five replicates ± the standard error (n = 5). Suijing18 groups: S1 = control, S2 = 45kg/hm² Si, S3 = 1 mM SA, S4 = 1 mM SA + 45kg/hm² Si; Hongke88 groups༚P1 = control, P2 = 45kg/hm² Si, P3 = 1 mM SA, P4 = 1 mM SA + 45kg/hm² Si; The sample was detected 10 days after spraying SA at booting stage.

3.3 The Agro-Morphology and Yield of the Dry-cultivated Rice

Combined SA + Si application most significantly increased the panicle number, grain number per panicle, and yield of the Suijing18 samples. The single-hole panicle number in S4 increased by 17.55% compared with S1, while the grain number per panicle increased by 26.92%. The 1000-grain weight in S4 increased by 2.1% compared with S1, while the setting rate was 15% higher. The output of S2, S3, and S4 increased by 62.34%, 53.48%, and 80.70% respectively (Table 2). The single-hole panicle number in the Hongke88 P4 sample increased by 29.27% compared with P1, while the number of grains per panicle increased by 22.59%. The 1000-grain weight of P4 increased by 10% compared with P1, while the setting rate was 1.19% lower. Compared with the P1 treatment, the yield of the P2, P3, and P4 treatments increased by 14.89%, 61.04%, and 77.26%, respectively. Compared with the P1 treatment, P4 significantly increased the number of single-hole panicles, grain number per panicle, 1000-grain weight, and yield of the rice samples (Table 2). Due to the weak drought resistance of Hongke88 and the strong drought resistance of Suijing18, the S1 yield was 40.5% higher than P1, while the P4 yield was 5.46% higher than S1 after combined SA + Si application.

Table 2

Effect of exogenous salicylic acid and silicon on yield and its components in drought cultivation rice.
Variety		Panicles per hill	Spikelets perpanicle	1000-grain weight (g)	Seeed-setting rate (%)	Yield (g/hill)
Suijing18	S1	13.33 ± 1.12b	60.67 ± 14.26b	21.59 ± 2.13a	0.78 ± 0.20b	12.64 ± 2.15c
	S2	13.00 ± 1.00b	75.44 ± 8.88a	22.72 ± 0.86a	0.92 ± 0.02a	20.52 ± 3.78ab
	S3	15.00 ± 2.29a	66.78 ± 13.05ab	23.22 ± 1.64a	0.86 ± 0.10ab	19.40 ± 2.67b
	S4	15.67 ± 0.69a	77.00 ± 11.42a	22.04 ± 1.24a	0.85 ± 0.13ab	22.84 ± 1.11a
Hongke88	P1	9.67 ± 1.00b	50.78 ± 1.31b	20.01 ± 1.32b	0.84 ± 0.02a	7.52 ± 2.30b
	P2	10.00 ± 0.89b	61.67 ± 5.92a	17.55 ± 0.68c	0.80 ± 0.03b	8.64 ± 1.89b
	P3	14.33 ± 0.50a	62.50 ± 5.74a	20.40 ± 0.53b	0.69 ± 0.04c	12.11 ± 0.57a
	P4	12.50 ± 0.55ab	62.25 ± 5.38a	22.01 ± 0.99a	0.83 ± 0.05ab	13.33 ± 0.26a

S1 = control, S2 = 45kg/hm² Si, S3 = 1 mM SA, S4 = 1 mM SA + 45kg/hm² Si ; P1 = control, P2 = 45kg/hm² Si, P3 = 1 mM SA, P4 = 1 mM SA + 45kg/hm² Si.The vertical bar indicates the means of three replicates (n = 5) and the error bar indicates the standard errors. Different letters indicate significant differences at p ≤ 0.05 (Tukey’s test).

3.4 The effect of combined SA + Si application on the chlorophyll fluorescence in the dry-cultivated rice

Chlorophyll fluorescence provides the most accurate response to the photosynthetic capacity, photosynthetic efficiency, and degree of environmental stress on plant leaves. The photosynthetic capacity and efficiency within dry-cultivated rice are low, but exogenous spraying of SA + Si can effectively reverse this unfavorable effect. Combined SA, Si, and SA + Si application restored the Fv/Fm values of the two dry-cultivated rice varieties to 0.7–0.85. This significantly improved the maximum PSII photochemical efficiency and enhanced the photosynthetic capacity of the rice cultivars, with SA + Si achieving the highest success. The Fv/Fm values of Suijing 18 S2, S3, and S4 increased by 14.71%, 28.33%, and 30.97%, respectively, compared to treatment S1, while the Fv/Fm values of Hongke 88 P2, P3, and P4 increased by 11.69%, 12.08%, and 19.17%, respectively, compared to P1 (Fig. 3a).The control groups of the two dry-cultivated rice varieties displayed low actual ΦPSII values and high NPQ values, which represented the response of the dry-cultivated rice to drought stress. However, the actual ΦPSII value increased after exogenous SA, Si, and SA + Si spraying. Compared with S1, the value in S4 increased by 29.01%, while that of P4 was 29.64% higher than P1 (Fig. 3b). Furthermore, the NPQ decreased after treatment. The value of S4 was 67.55% lower than S1, while that of P4 decreased by 36.98% compared with P1 (Fig. 3c). qP reflects the proportion of open PSII reaction centers, which is highly correlated with ΦPSII. The Suijing18 S2, S3, and S4 groups displayed lower values than S1, while those of the Hongke88 P2, P3, and P4 increased slightly compared with P1 (Fig. 3d). These results may be due to the variation in sensitivity to drought in the different dry-cultivated varieties.

3.5 Soluble Sugar and Soluble protein content

The presence of soluble sugar can improve the water absorption and retention capacity of plants and enhance the potential of cells to maintain water without interfering with normal metabolism, consequently improving the drought resistance of rice. Si, SA, and SA + Si were sprayed during the boot stage for sampling and testing at 2 h, 24 h, and 10 d, respectively. After SA + Si treatment, the soluble sugar content in the Suijing18 samples increased by 14.25% at 2 h and 10.72% at 10 d, respectively, compared with the control group, and decreased slightly at 24 h (Fig. 4a). However, a significant increase was evident in the Hongke88 samples. After SA + Si spraying, the soluble sugar levels began to increase after 24 h, which were 4.36%, 36.54%, and 32.22% higher in P2, P3, and P4, respectively, increasing to 26.5%, 52.63% and 61.58% after 10 d (Fig. 4b). After spraying, the soluble protein content in the Suijing18 samples rose substantially after 2 h, 24 h, and 10 d. The S2, S3, and S4 groups showed 27.96%, 15.35%, and 21.24% higher levels at 2 h, which increased to 30.11%, 17.47%, and 27.82% after 24 h, and 52.2%, 19.18%, and 17.42% after 10 d (Fig. 4c). SA + Si spraying elevated the soluble protein levels in the Hongke88 P2, P3, and P4 by 24.91%, 21.27%, and 30.91%, respectively, after 24 h, while these values were 27.49%, 18.23%, and 45.28% after 10 d (Fig. 4d). The SA + Si treatment groups of the two dry-cultivated rice varieties were horizontally compared. The results indicated that the soluble protein and soluble sugar levels were significantly higher in the Hongke88 samples than in Suijing18 at 24 h and 10 d. Therefore, combined SA + Si application substantially increased the soluble sugar and soluble protein content in the dry-cultivated rice, consequently improving its drought resistance.

3.6 The effect of combined SA + Si application on the antioxidant enzyme activity in the dry-cultivated rice

The two dry-cultivated rice varieties were sprayed during the booting stage and included the Control, Si, SA, and SA + Si treatment groups. The samples were monitored and assessed at 2 h, 24 h, and 10 d. The results showed that the SOD activity in the dry-cultivated rice samples were higher than in the control group at 24 h after Si, SA, and SA + Si spraying. This rise was more significant in the Suijing18 samples. S2, S3, and S4 were 36.35%, 42.74%, and 50.71%, respectively higher than S1 (Fig. 5a). Furthermore, P2, P3, and P4 were 3.59%, 20.31%, and 48.37%, respectively, higher than P1 (Fig. 5b). After 10 d of spraying, the detection trend was consistent with that at 24 h, indicating that exogenous SA + Si spraying optimally affected SOD activity at 24 h.

The POD activity in the Suijing18 S2, S3, and S4 treatment groups of Suijing18 was significantly higher than in S1 during the same period, showing a trend of S4 > S3 > S2 > S1 at 2 h, 24 h, and 10 d. At 24 h, the POD activity in the S2, S3, and S4 increased by 19.49%, 85.67%, and 114.33%, respectively, compared with S1 (Fig. 5c). The POD activity changes in the Hongke88 samples were consistent with those in Suijing18. At 24 h, the POD activity in P2, P3, and P4 increased by 20.79%, 23.64%, and 36.14%, respectively, compared with P1 (Fig. 5d). These results indicated that SA + Si treatment improved the antioxidant capacity of dry-cultivated rice by enhancing POD activity, and alleviated the damage caused by drought stress.

After Suijing18 sample treatment for 2 h, CAT activity trend was S2 > S4 > S3 > S1 after 2 h and S4 > S3 > S2 > S1 after 24 h and 10 d. Compared with S1, the CAT activity in S2, S3, and S4 increased by 11.93%, 21.62%, and 28.32%, respectively, after 24 h (Fig. 5e). The Hongke88 treatment groups yielded different results. After 2 h, the CAT activity was higher in P2, P3, and P4 than in P1, with P3 displaying the highest level of 200.30%. After 24 h, the P2 group exhibited the highest CAT activity at 107.76%, while the highest level was evident in the P4 group at 10 d, increasing by 6.33% (Fig. 5f).

The APX activity was higher in all the treatment groups at different detection times than in the control group. Drought stress usually induces H₂O₂ accumulation in rice. Higher APX activity can accelerate H₂O₂ removal and maintain the redox balance in cells, consequently improving the drought resistance of rice. At 24 h, the APX activity in S2, S3, and S4 increased by 40.90%, 64.84%, and 104.49%, respectively, compared with S1 (Fig. 5g), while this value was 40.90%, 64.84%, and 104.49% higher in P2, P3, and P4 than in P1 (Fig. 5h).

GR forms part of the antioxidant defense system and increases the drought resistance of rice at higher levels. The GR activity in the Suijing18 samples showed an order of S4 > S3 > S2 > S1 at 2 h and 10 d. At 24 h, this content was significantly higher in S4 and S2 than S1. At 2 h, 24 h, and 10 d, the respective GR levels in S4 increased by 55.00%, 18.18%, and 49.06% compared with S1 (Fig. 5i). The GR activity in Hongke88 P2, P3, and P4 treatment groups was higher than in P1. The four treatments showed an increasing trend of P4 > P3 > P2 > P1 during the same period. At 2 h, 24 h, and 10 d, the GR activity in P4 increased by 17.02%, 21.99%, and 70.43%, respectively, compared with P1 (Fig. 5j).

3.7 The effect of combined SA + Si application on the endogenous Si and SA content in the dry-cultivated rice

The Si content in the stems, leaves, and ears of the two dry-cultivated rice varieties were determined during the booting stage. The Si levels in the Suijing18 S2, S3, and S4 treatment groups increased compared to S1, while these values were also higher in the Hongke88 P2, P3, and P4 groups than in P1. The Si content in the stems, leaves, and ears of the two dry-cultivated rice cultivars was the highest after exogenous SA + Si spraying (Fig. 6a). However, differences were evident between the Si content in different treatment groups. Compared with S1, the Si content in S2, S3, and S4 in the stems increased by 158.97%, 496.42%, and 385.82%, respectively, while these values increased by 66.04%, 182.68%, and 218.05% in the leaves. Si content in S2, S3, and S4 in the ear increased by 160.72%, 570.51% and 542.8% (Fig. 6b). Compared with P1, the Si content in the P2, P3, and P4 treatment groups increased by 77.73%, 70.01%, and 516.22% in the stems, 65.21%, 56.67%, and 351.66% in the leaves, and 54.95%, 45.10%, and 371.15% in the panicles (Fig. 6c). In addition, the SA content in the different treatment groups of the two dry-cultivated rice varieties was recorded at 2 h, 24 h, and 10 d. The results indicated that the SA levels in the Suijing18 S2, S3, and S4 treatment groups increased by 6.64%, 25.78%, and 42.02%, respectively, compared to S1 at 2 h. At 24 h, these levels in the S2, S3, and S4 groups increased by 8.90%, 22.70%, and 45.01%, respectively, compared with S1, and were 9.67% and 27.70% higher at 10d in the S3 and S4 groups. The SA content in the S2 group was lower than in S1 (Fig. 6d). At 2 h, the SA content in the Hongke88 P2, P3, and P4 groups were 1.54%, 24.60%, and 29.98% higher than in P1. At 24 h, these values were 10.22% and 33.26% higher in the P3 and P4 groups than in P1. At 10 d, the SA content in the P3 and P4 groups increased by 16.74% and 29.21% compared with P1, respectively, while this value was lower in the P2 group at 24 h and 10 d than in P1 (Fig. 6e).

Rice has different mechanisms to cope with drought, such as the activation of enzymatic and nonenzymatic antioxidant systems and the accumulation of compatible osmolytes.²⁵ Drought stress affects rice osmoregulators such as soluble sugars, which are key osmoprotectors in plants. Osmoregulators maintain and protect plant macromolecules and structures from stress damage and increase their tolerance to drought stress.²⁶ Drought hinders carbon fixation into sugars via photosynthesis and affects their transport by decreasing the cellular osmotic potential, reprogramming the cellular and subcellular sugar distribution.²⁷ In addition, drought tolerance in crops is associated with higher protein content, and the concentration of soluble proteins depends on the nature of the plant species and the type of tissue under water stress.²⁸ Studies have shown that soluble sugar and soluble protein accumulation in rice enhances its drought tolerance adaptation.²⁹ Therefore, increasing osmoregulatory substance accumulation in rice is essential to enhance its drought tolerance and has attracted significant research attention. The exogenous application of natural endogenous, bioactive compounds (e.g., SA) may be an alternative approach to increase crop productivity.³⁰ SA is considered as an environmentally friendly and suitable chemical regulator.³¹ The use of SA in drought-stressed plants improves of osmoregulator functionality in plants.³² Our previous research showed that Si maintained normal physiological and biochemical metabolism in rice in drought conditions by regulating soluble sugars, which increased the cytoplasmic concentration and decreased its osmotic potential. This helped to maintain water uptake and cellular expansion, which ensured sustained rice growth in drought conditions and enhanced its adaptability.³³ Considering the foundation of the previous work, the current study used combined SA + Si application to enhance the drought tolerance and yield of dry-cultivated rice both above and below the ground. Lawan Gana et al. found that individual SA and Si application increased the soluble sugar and soluble protein accumulation in rice seedlings.³⁴ AliSu Yang et al. revealed that the synergistic effect of SA and Si enhanced the accumulation of soluble sugars and soluble proteins, consequently reducing chromium toxicity in rice.³⁵ In the current study, the combined application of SA + Si significantly increased the soluble sugar content in the Suijing18 and Hongke88 rice cultivars (Figs. 4a and 4b). The soluble sugar content in Suijing18 samples increased by 14.25% at 2 h compared to the control, while the soluble sugar content displayed an initial decline at 24 h, followed by a rise at 10 d, increasing by 10.72% compared to the control (Fig. 4a). Although the soluble sugar content was low in the Hongke88 samples at 2 h, it increased by 32.22% at 24 h and 61.58% at 10 d compared with the control (Fig. 4b). The soluble protein levels were higher in both the Suijing18 and Hongke88 treatment groups than in the control (Figs. 4c and 4d) after exogenous SA + Si spraying. The Hongke88 variety displays weak drought-resistance while the Suijing18 cultivar exhibits strong tolerance to drought. After exogenous SA + Si spraying, the Hongke88 samples displayed higher soluble sugar and soluble protein levels than the Suijng18 groups at the same detection times and exogenous spraying concentrations. This study demonstrated for the first time that exogenous SA + Si significantly increased the soluble sugar and soluble protein content in dry-cultivated rice, consequently enhancing its drought resistance. This treatment was more effective in the dry-cultivated rice showing weak drought tolerance.

A strong root system facilitates the uptake of water and nutrients from the soil by the crop and promotes its growth.³⁶ When rice suffers from drought stress, the root system first senses the lack of water in the external environment via changes in water potential, osmotic pressure, and other physical properties, which restricts root system growth.³⁷ The present study showed that exogenous SA + Si significantly increased the root length, plant height, aboveground dry weight, and belowground dry weight of two dry-cultivated rice cultivars. Moreover, combined SA + Si application was more effective than these substances alone (Table 1 and Fig. 2) to increase in the root size and drought tolerance of the rice plants, resulting in higher yields.³⁸ The yield of the Suijing18 S4 group was 80.70% higher than S1, while the yield of the Hongke88 P4 group was 77.26% higher than P1 (Table 2), which were consistent with the results of previous studies.

Drought triggers multiple morphological, physiological, and metabolic changes in plants,⁸ stimulating the production of ROS. To counteract the effect of ROS, rice plants develop antioxidant enzymes such as SOD, POD, CAT, APX, and GR, which scavenge stress factors such as salinity, drought, and extreme-temperature excess ROS.³⁹ SOD is the main line of defense against drought-induced oxidative stress⁸ while the superoxide (O₂ ^⦁−) produced by the mitochondrial electron transport chain (ETC) or RBOH is converted to H₂O by SOD.⁴⁰ CAT is a key enzymes in the biological defense system during the biological evolution process. It reduces H₂O₂ overproduction during oxidative stress by converting H₂O₂ to H₂O and O₂, while playing a vital role in plant defense and stress response, delaying plant senescence, and regulating plant cell redox balance.⁴¹ The ascorbate-GSH pathway represents the main HO₂₂ detoxification system in plant cells, in which APX is an effective ROS regulator since it contributes the most to H₂O₂ detoxification, utilizing ascorbic acid as an electron donor to convert H₂O₂ to water.^{42 43} GR is a highly conserved enzyme that combines NADPH oxidation and GSH disulfide (GSSG) reduction to decrease GSH, consequently eliminating excessive ROS.⁴⁴ Studies have shown that enhancing the activities of ROS-scavenging enzymes such as POD, CAT, APX, GPX, and GR in rice can regulate its drought tolerance.⁴⁵ Exogenous SA application induces SA-binding protein 2 gene overexpression, which plays a positive role in regulating the expression of antioxidant enzyme-related genes and activities in tobacco plants, Foliar sprays with appropriate SA concentrations increases the SOD levels and POD activities in leaves, protecting the photosynthesis process.¹⁵ The combined application of SA and Si to the aboveground section of Scrophularia striata L. improves its response to drought stress by reducing the MDA and H₂O₂ content.⁴⁶ In this study, the combined SA + Si application significantly enhanced the antioxidant enzymes in the two dry-cultivated rice species (Fig. 5), effectively improving their antioxidant capacity and drought tolerance.⁴⁵ In addition, exogenous SA application restored the chlorophyll content and CAT activity in the rice, and further increased the activity of other enzymatic antioxidants to enhance drought resistance.³² Therefore, combined SA + Si application significantly increased the SA and Si levels in the two dry-cultivated rice cultivars (Fig. 6), enhancing their antioxidant defense capacity, reducing the degree of damage caused by drought, and improving crop yield.

In recent decades, solar-induced chlorophyll fluorescence (SIF) has provided a unique method to estimate photosynthetic functional traits in crops.⁴⁷ Light energy captured by chlorophyll molecules is used in photochemical reactions to drive photosynthesis, dissipating excess light energy as heat (non-photochemical burst, NPQ) or emitting it as chlorophyll fluorescence. These processes compete with each other, e.g., more significant photosynthesis decreases the dissipation of the additional energy. Therefore, by measuring the chlorophyll fluorescence yield, it is also possible to estimate the efficiency of the photochemical reactions and the heat dissipation. Membrane permeability and vesicle function in chloroplasts are impaired when plant cells are subjected to adversity stress, causing a gradual diminution of photosystem activity and chlorophyll fluorescence. This affects photosystem II repair in plants in adverse conditions, enhances photoinhibition, and reduces photosynthetic efficiency.⁴⁸ The chlorophyll fluorescence indexes include the Fv/Fm value, ΦPSII, NPQ, and qP. The chlorophyll content and fluorescence parameters reflect the response of total photosynthetic productivity to environmental factors. Flag leaves represent the most important organ source of rice, playing a crucial role in supplying assimilates for seed grain development.⁴⁹ This study investigated the genetic factors controlling chlorophyll content and fluorescence parameters in rice flag leaves. The Fv/Fm ratios were significantly higher in the two dry-cultivated rice treatment groups than in the control groups, indicating that the functional state of the chlorophyll in these rice cultivars was significantly restored after exogenous SA + Si spraying⁵⁰ (Fig. 3a). The ΦPSII of the two dry-cultivated rice species was also substantially enhanced compared with the control, indicating that exogenous SA + Si spraying improved the energy conversion efficiency of chloroplasts during photosynthesis⁵¹ (Fig. 3b). The NPQ responds to the regulatory mechanisms in plants under stress, dissipating excess light energy in various ways.⁵² In this study, the NPQ values of the two dry-cultivated rice species were lower than in the control group, especially that of Suijing18, indicating that exogenous SA + Si spraying significantly enhanced their drought tolerance (Fig. 3c). The qP is the photochemical burst coefficient based on the swamp model in which the PSII reaction centers are connected by a common antenna. The qP reflects the portion of light energy absorbed by the pigments in the PSII antennae that can be used for photochemical electron transfer.⁵³ The qP value decreased in the Suijing18 cultivar, while it displayed a slight decrease in the Hongke88 variety (Fig. 3d). These results indicated that different drought-resistant cultivars showed varied sensitivity to drought, inducing different qP responses in vivo.

The combined application of SA and Si improves the drought resistance of two kinds of dry-cultivated rice by increasing the content of osmoregulatory substances, enhancing the levels of endogenous SA and Si, upregulating the activity of antioxidant enzymes, and strengthening the photosynthetic capacity of the PSII system. This results in improved drought-resistant capability and increase of the accumulation of dry matter and yield of dry-cultivated rice.

Author Contribution

GW :Writing – review & editing, Writing – original draf, Funding acquisition. Y W：Draw figures1-2, Q L:Draw figures3-4, XS:Draw figures5-6, AS: Draw tables1-2, HJ:Investigation, XM:Investigation, XW:Investigation, MY:Review manuscript, ZW:Review manuscript, Funding acquisition .All the authors reviewed the manuscript.

Data Availability

All data for dry-cultivated rice that support the results of this study are included within this paper and its supplementary information files

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No competing interests reported.

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Combined action of salicylic acid and silicon regulates osmoregulatory substances and antioxidant enzymes to improve drought tolerance and yield in dry-cultivated rice

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Abstract

Figures

1. Introduction

2. Materials and Methods