Foliar application of silicon improves growth of soybean by enhancing carbon metabolism under shading conditions

Plant Physiology and Biochemistry 159 (2021) 43–52

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Foliar application of silicon improves growth of soybean by enhancing carbon metabolism under shading conditions Sajad Hussain a, b, Maryam Mumtaz a, Sumaira Manzoor c, Li Shuxian a, b, Irshan Ahmed a, b, Milan Skalicky d, Marian Brestic d, e, Anshu Rastogi f, Zaid Ulhassan g, Iram Shafiq a, b, Suleyman I. Allakhverdiev h, Haris Khurshid i, Wenyu Yang a, b, **, Weiguo Liu a, b, * a

College of Agronomy, Sichuan Agricultural University, 211-Huimin Road, Wenjiang District, Chengdu, 611130, PR China Sichuan Engineering Research Center for Crop Strip Intercropping System, Key Laboratory of Crop Ecophysiology and Farming System in Southwest China (Ministry of Agriculture), Sichuan Agricultural University, Chengdu, PR China c Department of Botany, Government College University of Faisalabad, Layyah Campus, 31200, Pakistan d Department of Botany and Plant Physiology, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, 16500, Prague, Czech Republic e Department of Plant Physiology, Slovak University of Agriculture, 94976, Nitra, Slovakia f Laboratory of Bioclimatology, Department of Ecology and Environmental Protection, Poznan University of Life Sciences, Piątkowska 94, 60-649, Poznan, Poland g Institute of Crop Science, Ministry of Agriculture and Rural Affairs Laboratory of Spectroscopy Sensing, Zhejiang University, Hangzhou, 310058, China h К.А. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya St. 35, Moscow 127276, Russia i Oil Seed Research Program, National Agricultural Research Centre, Islamabad, Pakistan b

A R T I C L E I N F O

A B S T R A C T

Keywords: Silicon Photosynthesis Shade stress Soluble sugar

An experiment was set up to investigate physiological responses of soybeans to silicon (Si) under normal light and shade conditions. Two soybean varieties, Nandou 12 (shade resistant), and Nan 032–4 (shade susceptible), were tested. Our results revealed that under shading, the net assimilation rate and the plant growth were significantly reduced. However, foliar application of Si under normal light and shading significantly improved the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and decreased intercellular carbon dioxide concentration (Ci). The net photosynthetic rate of Nandou 12 under normal light and shading increased by 46.4% and 33.3% respectively with Si treatment (200 mg/kg) compared to controls. Si application also enhanced chlorophyll content, soluble sugars, fresh weight, root length, root surface area, root volume, rootshoot ratio, and root dry weight under both conditions. Si application significantly increased the accumulation of some carbohydrates such as soluble sugar and sucrose in stems and leaves ensuring better stem strength under both conditions. Si application significantly increased the yield by increasing the number of effective pods per plant, the number of beans per plant and the weight of beans per plant. After Si treatment, the yield increased 24.5% under mono-cropping, and 17.41% under intercropping. Thus, Si is very effective in alleviating the stress effects of shading in intercropped soybeans by increasing the photosynthetic efficiency and lodging resistance.

1. Introduction The potential of silicon (Si) for alleviating the effects of biotic and abiotic stress in plants is well known (Frew et al., 2018). Silicon plays a vital role in counteracting abiotic stresses such as nutrient imbalance, water deficit, temperature, shade and physical stresses (Helaly et al., 2017; Tripathi et al., 2016b). Silicon belongs to the IVA group in the periodic table and is the second most abundant element in soils (Sommer

et al., 2006), but its availability to plants as silicic acid could be limiting, hence the application of silicate fertilizers is required in crop production (Ma and Takahashi, 2002). In soil solution, it is present as a silicic acid with a concentration of 0.1–0.6 mM. However, most Si exists in soil combined with oxygen to form oxides and silicates, which are not easily taken up by plants. On the agronomic basis, monosilicic acid phase of Si is essential. Plants take up silicon in the form of silicic acid and farmers use monosilicic adic, Si(OH)4, to provide crops with Si. Several studies

* Corresponding author. College of Agronomy, Sichuan Agricultural University, 211-Huimin Road, Wenjiang District, Chengdu, 611130, PR China. ** Corresponding author. College of Agronomy, Sichuan Agricultural University, 211-Huimin Road, Wenjiang District, Chengdu, 611130, PR China. E-mail addresses: [email protected] (S. Hussain), [email protected] (W. Yang), [email protected] (W. Liu). https://doi.org/10.1016/j.plaphy.2020.11.053 Received 11 August 2020; Accepted 29 November 2020 Available online 5 December 2020 0981-9428/© 2020 Elsevier Masson SAS. All rights reserved.

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Plant Physiology and Biochemistry 159 (2021) 43–52

have shown the beneficial effects of Si application in terms of faster growth and increased yield and improved uptake of macro- and micro-nutrients (Ma and Takahashi, 1990; Tripathi et al., 2016b). Si is not considered as an essential element for plants, although recent studies have been debating whether that is still true. It has been the subject of global attention as it can enhance a plant’s resistance to disease and herbivorous insects, thus reducing the use of fungicides and pesticides. Si is also now considered as an environment-friendly element (Jian and Eiichi, 2002). The effects of Si vary among plant species, but in general it enhances instantaneous water-use efficiency and photosyn­ thesis, as well as remedying nutrient imbalances (Farooq et al., 2013; Feng et al., 2010). Plants have a well-developed defense system against oxidative damage, including antioxidant enzymes and compounds (Hussain et al., 2020b). The enzymatic antioxidant activities decreased under stress from metal contamination and silicon enhanced the activity of superoxide dismutase, peroxidases, catalase, and ascorbate peroxi­ dase, which help plants cope with metal stress (Adrees et al., 2015). Si application increased the activities of non-enzymatic antioxidants like glutathione, non-protein thiols and ascorbic acid in rice, cucumber and pak choi under Mn and Cd stress (Song et al., 2009; Wang et al., 2015). The addition of Si had minimal effects on photosynthesis and chloro­ phyll fluorescence in tomato and maize plants under normal conditions, but under stress, Si improved photosynthesis and chlorophyll fluores­ cence yields (Al-aghabary et al., 2005). Si application on agricultural crops increased photosynthesis and enzymatic activities resulting in higher yield and increased biomass. Si increased the chlorophyll pig­ ments in maize, wheat, soybean and other plant species and foliar application of Si significantly enhanced PSII efficiency (Tripathi et al., 2003). The uptake of N, P, Ca and Mg by plant were observed to be also increased after Si application. Silicon application was reported to have beneficial effects on plant growth as it enhanced resistance to lodging, biotic and abiotic stress, and had indirect effects such as pH adjustment and assimilation of macroand micronutrients contained in silicate fertilizers. Si application improved biomass production (Mehrabanjoubani et al., 2015; Vaculík et al., 2009), yield and quality of a wide range of crops including monocots like rice, wheat, maize, barley, millet, sorghum and sugarcane that actively take up and accumulate large amounts of Si in their organs, and some dicots like cotton, vegetable and fruit crops (Liang et al., 2015a). Earlier findings suggested a positive role of Si in enhancing stem length, leaf area, photosynthesis, stomatal conductance, transpiration rate, stomatal number and size, pigment concentration and chlorophyll fluorescence under stress conditions (Abbas et al., 2015; Mateos-Naranjo et al., 2013; Shen et al., 2010a, 2010b, 2014; Xiaoqin et al., 2011). Shade is a pervasive abiotic stress in agricultural crop production especially in intercropping (Hussain et al., 2020b; Yang et al., 2018). When soybeans are intercropped with maize, they suffer shading from the adjacent maize canopy. Shade leads to stem and petiole elongation and increased internodal distance, and reduces root diameter and stem strength (Hussain et al., 2019d; Wei-Guo et al., 2018; Wen et al., 2020). Shading makes soybean plants susceptible to lodging thereby inhibiting the transport of photo-assimilates, nutrients, and water (Li et al., 2014), which reduces mechanized yield (mechanical harvesting efficiency) (Hussain et al., 2019b; Wei-Guo et al., 2018). It affects the rate of photosynthesis by blocking electron transport from photosystem II to photosystem I, thus reducing the electron transport rate, the amount of ATP produced, and rubisco activity (Huang et al., 2018; Hussain et al., 2019a; Valladares and Niinemets, 2008; Yao et al., 2017). In addition, shading decreases quantum yield, effective quantum yield of photo­ system, photochemical quenching (Hussain et al., 2019c), and electron transport rate (Hussain et al., 2019a). Shade is also known to reduce nonstructural carbohydrates such as soluble sugars, starch and sucrose, which not only provide energy but also contribute to the accumulation of structural carbohydrates (Wu et al., 2017). In the literature, there have been many reports about the positive role of Si in plant growth under conditions of salt stress, metal toxicity,

drought stress, radiation damage, nutrient imbalances, high tempera­ ture, and freezing (Ma et al., 2002). However, not much is known about the effect of Si on soybean plants under shade conditions. Therefore, the current study was aimed to investigate the role of Si in the improvement of morphological characteristics, photosynthetic parameters, and non-structural carbohydrates in soybeans grown under shade. The effect of Si on various yield parameters of soybean was also studied. 2. Materials and methods The experiment was conducted at Sichuan Agricultural University, Wenjiang district, Chengdu, Sichuan province in 2018. The experi­ mental design was completely randomized using soybean cultivars, Nandou 032–4 (shade susceptible) and Nandou 12 (shade resistant). Three to five seeds were sown in plant pots 27 cm in diameter and 18 cm high and water was added to maintain soil capacity. The soil was a loam with pH 6.7, total nitrogen 2.1 g kg− 1, magnesium 21.3 g kg− 1, available phosphorus 24.5 mg/kg, available potassium 121 mg/kg, and available nitrogen 130 mg/kg. Each treatment consisted of five pots with three replications. Four days after seedling emergence, seedlings were trans­ ferred to light and shade conditions. The shading condition (356 μmolm− 2s− 1) was provided by black shade net that allowed 28% of full sunlight. The light condition was provided by placing the plants in full sunlight (1189 μmolm− 2s− 1). Intensity of photosynthetically active ra­ diation (PAR) was measured by 138 HR350 (Hipoint Inc., Gaoxiong, Taiwan). The Si treatments consisted of CK: 0 mg/L; S1: 100 mg/L; S2: 200 mg/L, S3: 300 mg/L both cropping conditions. The silicon source was Na2SiO3.9H2O applied through a foliar spray, and each treatment was repeated three times. The first foliar treatments were applied when the true leaves unfolded, and treatments were done every 7–10 days thereafter, for a total of two times. The field experiment for soybean yield was carried out at the Renshou experimental site, Sichuan Agricultural University in Chengdu, Sichuan province. The average annual temperature, rainfall and sun­ shine duration were 17.4 ◦ C, 1009.4 mm and 1196.6 h, respectively. The test site contained purple soil with uniform fertility. Randomized com­ plete block design with factorial arrangement was used for layout, the two factors were maize-soybean intercropping pattern (intercropping and monocropping), and the Si application. In the intercropping pattern, maize (Chuandan 418) was planted in wide (160 cm apart) and narrow (40 cm apart) rows, whereas plant to plant spacing was 33.5 cm. Soy­ bean was sown in wide rows of maize. The row to row and plant to plant spacing of soybean were 40 cm and 20 cm, respectively. In monocrop­ ping system, soybean was planted with row to row distance of 50 cm and plant to plant distance of 20 cm. The plot size was (6 m2) including three strips of soybean (6 rows) in intercropping and monoculture. 2.1. Morphological and growth measurements 2.1.1. Agronomic traits For agronomic measurements the soybean seedlings were grown for 35–45 days. The stems and leaves from nine plants were weighed separately for each treatment at 4th trifoliate stage, heated at 105 ◦ C for 1 h and dried to constant weight at 75 ◦ C to determine biomass. A simple electronic balance was used to weigh the samples. Plant height was measured with a meter stick and stem diameter with a Vernier caliper. 2.1.2. Snapping resistance Snapping resistance was determined using a digital force tester (YYD1, Zhejiang Pu Instrument Co. LTD, Hangzhou, China). 2.1.3. Root morphology assessment Soybean plants were uprooted gently and placed in running water to remove the soil. After washing the roots carefully, they were spread on a transparent Plexiglas tray and covered with 2–3 mm of water. The washed roots contained very small and short pieces that could not be 44

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Fig. 1. Effect of Si on plant height (A), internode length (B), stem diameter (C) and snapping resistance (D) of Nandou 12 and Nandou 032–4 under normal light and shade conditions. Values are the mean of three replicates. Bars indicate ±SD. Lowercase letters above the bars indicate significant difference (p < 0.05) between treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

45

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Fig. 2. Effect of Si on photosynthetic rate (A), stomatal conductance (B), transpiration rate (C) and intercellular carbon dioxide (D) of Nandou 12 and Nandou 032–4 under normal light and shade conditions. Values are the mean of three replicates. Bars indicate ±SD. Lowercase letters above the bars indicate significant difference (p < 0.05) between treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

46

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seen by image filtering. The tray containing the roots was placed on an Epson Perfection V700 photo scanner and scanned. Afterwards, WinR­ HIZO (Version, 2007d; Regent Instrument Inc., Canada) software was used to determine the total root length, surface area, root volume and diameter (Hussain et al., 2019a).

3.1.2. Effect of silicon application on internode length Under normal light, after Si application, the internode length of the first stem of Nandou 12 increased significantly at 100 mg/kg and 300 mg/kg, but did not change significantly at 200 mg/kg (Fig. 1B). In the shade, after Si application, the internode length was significantly reduced at 100 mg/kg and 300 mg/kg Si. Length was slightly reduced at 200 mg/kg, but the difference was not significant. The internode length of the first internode of Nandou 032–4 was under normal light. After silicon treatment, the internode length was significantly reduced compared to CK, and the change was significant with 300 mg/kg treatment. After shading, addition of silicon had no significant effect on internode length.

2.1.4. Chlorophyll contents Chlorophylls and carotenoids were extracted with 80% acetone and measured according to (Lichtenthaler, 1987). 2.1.5. Photosynthetic characteristics All measurements of net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr), were made using an LI-6400 portable photosynthetic assay system (LI-6400-09; LiCor, Lincoln, Nebraska,USA) and taken on sunny day from 10:00 to 11:30 a.m. Manual control conditions were CO2 concen­ tration 400 μmoL mL− 1 and light intensity 450 μmoLm− 2s− 1 (Iqbal et al., 2019).

3.1.3. Effect of silicon application on stem diameter Under normal sunlight, the first stem thickness of the soybean vari­ eties increased significantly after Si treatment. With increasing Si con­ centration, the stem thickness first increased and then decreased under normal light and shade conditions. The maximum value of Nandou 12 was reached at 200 mg/kg Si, an increase of 17.5% compared to CK. Under normal light, the stem diameter of the first internode of Nandou 032–4 increased by 14.2% than CK at 100 mg/kg and then decreased with increasing Si concentration. Under shade, the variation in stem diameter was the same as under normal light, and the maximum value was recorded at 100 mg/kg, which was 18.2% higher than CK (Fig. 1C).

2.1.6. Carbohydrate analysis Stems were dried to a constant weight, crushed, and sieved through an 80 mm mesh sieve (Hussain et al., 2020a). Samples of 0.15 g of the powder were placed in 50 mL centrifuge tubes, 1.5 ml H2SO4 (72%) was added and tubes were placed in a shaking water bath at 30 ◦ C for 1 h. A volume of 42 mL of deionized water was then added to each tube. After homogenization, the paste was hydrolyzed in a high-temperature ster­ ilizer at 121 ◦ C for 2 h. The supernatant was centrifuged and filtered through a 0.22 μm micro-porous membrane. The composition was determined by high performance liquid chromatography (Agilent HPLC 1100, Agilent, USA) with the following conditions: Amine x < HPX-87H Column C (300 × 7.8 mm), RID detector, ultra-pure water as mobile phase, column temperature 55 ◦ C, RID temperature 50 ◦ C, injection volume 10 μL, and flow rate 0.6 mL/min.

3.1.4. Effect of silicon application on stem snapping resistance As can be seen for the pot experiment in Fig. 1D, there is a significant difference between shade-grown and normal light-grown plants in the basic snapping resistance of the soybean stems. Under normal light and shade conditions, the control plant without any exogenous Si treatment (CK) was significantly different from other treatments. Under normal light, the snapping resistance of Nandou 12 increased significantly after Si treatment, reaching its highest value at 200 mg/kg, a 73.7% increase compared to CK. Under shade, the stem snapping resistance first increased and then decreased with increasing Si, reaching a maximum at 100 mg/kg, 47.8% higher than CK. In Nandou 032–4, the stem snapping resistance first increased and then decreased with increasing Si under normal light and shade conditions, reaching a maximum at 200 mg/kg, for an increase of 34.2% and 35.0% respectively compared to CK. Thus, under normal light and shade conditions, suitable applications of silicon can increase the mechanical strength, promote growth, and enhance resistance to lodging of soybean stems.

2.2. Yield determination At the maturity stage, 10 plants were selected from the investigation plot to measure plant height, number of pods, number of effective branches, seed number per plant, seed weight per plant, and 100-seed weight. 2.3. Statistical analysis

3.1.5. Effect of silicon application on photosynthetic parameters The net photosynthetic rate (Pn) of Nandou 12 increased signifi­ cantly after Si application under normal light or shade conditions (Fig. 2A). The maximum net photosynthetic rate under normal light (46.4%) and shade (33.3%) was occurred after 200 mg/kg Si. The net photosynthetic rate of Nandou 032–4 also significantly increased after Si treatment under normal light by 52.9% and shade by 13.8% compared to the control (CK). The stomatal conductance (Gs) of Nandou 12 increased significantly after application of silicon (100 and 200 mg/kg) under normal light and shade (Fig. 2B). Under shade, stomatal conductance of Nandou 12 increased by 44.0% than CK at 300 mg/kg of Si. However, Nandou 032-4 had highest stomatal conductance at 100–200 mg/kg of Si application under both conditions (normal light and shade). Foliar application of Si significantly increased the transpiration rate (Tr) of Nandou 12 under normal light and shade conditions (Fig. 2C). Under normal light, 200 mg/kg Si increased the Tr by 60.4% as compared to CK. Under shade, however, the trend of Nandou 032–4 was different from that of Nandou 12. After 100 mg/kg treatment, the transpiration rate of Nandou 032–4 increased, but the difference was not significant. After 200 mg/kg and 300 mg/kg treatment, it decreased significantly. As shown in Fig. 2D, the intercellular carbon dioxide concentration

The data for each trait was analyzed using SPSS. An analysis of variance (ANOVA) test was used to determine the treatment effects on the measured variables. The least significance difference (LSD) test was performed to compare means at 5% probability level. SigmaPlot was used for graphical presentation of data. 3. Results 3.1. Effect of silicon application on morphological characteristics of soybean varieties 3.1.1. Effect of silicon application on plant height As can be seen from Fig. 1A, the height of Nandou 12 increased by 105.0% and Nandou 032-4 by 61.0% under shade S0 compared to CK. This is a typical shade avoidance reaction. Spraying Si on the leaf surface under normal light had no significant effect on the height of the two soybean varieties. In the shade, however, Si application significantly reduced the height of Nandou 12, but the difference between the three Si levels was not significant. For Nandou 032–4, the height was signifi­ cantly reduced at 300 mg/kg Si, while the difference between the other two Si levels was not significant. 47

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Table 1 Effect of silicon on morphological characteristics of soybean roots. Cultivar

Nandou 12

Si (mg/kg)

Light

CK 100 200 300

Shade

CK 100 200 300

Nandou 032-4

Light

CK 100 200 300

Shade

CK 100 200 300

Length (cm)

Table 2 Effect of Si on stem lodging resistance index.

Surface area (cm2)

Avg diameter (mm)

Root volume (cm3)

1478.92 b 2333.13 ab 2546.66 a 3344.45 a

174.08 b

0.450 a

1.42 b

298.78 a

0.456 a

2.89 a

308.80 a

0.439 a

2.77 a

421.19 a

0.437 a

4.03 a

1395.19 b 2485.22 a 2589.54 a 2436.74 a

132.34 b

0.407 b

0.66 b

262.00 a

0.435 a

2.25 a

257.68 a

0.425 a

2.10 a

275.80 a

0.419 ab

2.22 a

2421.67 b 2107.51 b 2473.25 b 3641.92 a

250.14 ab

0.392 a

1.72 d

199.06 b

0.436 a

2.93 b

273.47 ab

0.413 a

2.14 c

394.50 a

0.388 a

3.08 a

1606.02 b 2759.94 a 2227.47 ab 2336.69 ab

167.94 b

0.422 a

1.12 b

294.04 a

0.395 a

2.17 a

249.10 ab

0.419 a

1.96 ab

259.38 ab

0.416 a

2.02 ab

Si (mg/kg) CK 100 200 300

Nandou 12

Nandou 032-4

Light

Shade

Light

Shade

2.63 d 3.16 c 4.16 b 4.82 a

1.43 c 2.35 bc 2.90 a 2.60 ab

3.84 c 4.95 a 4.36 b 3.70 d

1.84 d 2.66 c 2.70 a 2.69 b

Note: Values followed by a different small letter indicate significant difference (p < 0.05). Table 3 Effect of Si on soluble sugar content (SSC) in soybeans under normal light or shade. Cultivar

Si (mg/kg)

Leaves

Soluble Sugars (mg/g) Stems

Roots

Nandou 12

Light

CK 100 200 300

31.19 c 48.36 a 46.46 a 33.54 b

142.43 a 112.41 b 106.70 c 91.69 d

101.95 a 61.67 b 50.84 c 28.22 d

Shade

CK 100 200 300

25.59 c 45.32 a 45.13 a 40.19 b

90.93 b 106.13 a 74.02 d 84.85 c

40.00 16.63 16.25 28.98

a b b ab

Light

CK 100 200 300

29.17 d 37.84 b 51.41 a 34.11 c

102.33 b 101.95 b 112.41 a 96.06 c

18.34 11.50 33.73 21.95

c d a b

Shade

CK 100 200 300

20.88 c 28.98 b 28.79 b 40.57 a

73.07 d 96.06 b 82.57 c 126.09 a

26.13 32.78 32.97 36.58

b a a a

Nandou 032-4

Means that do not share the same letters in the column differ significantly at p < 0.05.

Note: Values followed by a different small letter indicate significant difference (p < 0.05); the same below.

the lodging rate (Table 2).

(Ci) in Nandou 12 was significantly reduced after exogenous Si appli­ cation under normal light and shade conditions. The order of Ci reduction was CK > 300 mg/kg > 100 mg/kg > 200 mg/kg, and the change was greatest at 200 mg/kg Si, which decreased Ci by 42.8% in normal light and 35.9% in shade condition compared to CK. However, under shading, a sharp decrease in Ci was observed in Nandou 032–4 at 300 mg/kg Si. Under normal light, the Ci of Nandou 032–4 increased significantly at 300 mg/kg.

3.1.8. Effects of silicon on carbohydrate content of leaves, stems, and roots 3.1.8.1. Soluble sugar content (SSC). Table 3 shows that, in the pot experiment, the soluble sugar content (SSC) in the leaves and stems of Nandou 12 and Nandou 032–4 was significantly reduced by shading, Table 4 Effect of Si on starch content in soybean cultivars under normal light and shade conditions.

3.1.6. Effect of silicon application on root morphology Table 1 shows that under normal light and shade conditions, the length, surface area, and volume of roots of Nandou 12 treated with silicon were significantly greater than CK, and there was no significant difference between Si concentrations. The length, surface area and volume of Nandou 032–4 roots increased to different degrees after different Si treatments under normal light and shade conditions, while the root thickness did not change significantly under different light conditions. We conclude that Si application can increase root growth of shaded soybeans. 3.1.7. Effect of silicon application on lodging The lodging resistance indices of Nandou 12 and Nandou 032–4 were significantly increased by Si before and after harvest. Nandou 12 had highest lodging resistance index with the Si application 300 and 200 mg/kg under light and shade conditions respectively. However, Nandou 032-4 had highest lodging resistance index with the Si application of 100 and 200 mg/kg under light and shade condition respectively, and were highest with 200 mg/kg and 300 mg/kg Si treatment. Thus, Si appli­ cation can improve the lodging resistance index of soybeans and reduce

Cultivar

Si (mg/kg)

Leaves

Starch Content (mg/g) Stems

Roots

Nandou 12

Light

CK 100 200 300

128.02 a 80.47 b 80.98 b 33.95 c

39.25 a 34.63 b 36.08 a 20.10 c

30.70 a 26.43 b 30.02 a 31.90 a

Shade

CK 100 200 300

69.87 b 80.47 a 59.43 c 50.20 d

20.61 c 24.03 b 30.95 a 31.21 a

21.64 b 25.23 a 19.93 b 24.71 a

Light

CK 100 200 300

115.02 a 35.15 c 85.77 b 117.93 a

30.19 b 28.65 b 34.89 a 31.21 b

20.10 d 25.23 c 29.50 b 39.77 a

Shade

CK 100 200 300

75.17 a 70.89 b 60.80 c 43.70 d

25.83 b 27.96 b 36.43 a 20.44 c

39.77 a 35.49 b 34.81 bc 32.92 c

Nandou 032-4

Means that do not share the same letters in the column differ significantly at p ≤ 0.05. 48

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Table 5 Effect of Si on sucrose content in soybean cultivars under normal light and shade (mg⋅g− 1). Cultivar

Si (mg/kg)

Leaves

Stems

Roots

Nandou 12

Light

CK 100 200 300

17.21 31.30 21.17 15.98

c a b c

53.54 b 57.23 a 53.54 b 35.99 c

20.43 36.98 15.48 22.40

b a c b

Shade

CK 100 200 300

26.36 21.66 38.22 17.95

b ab a c

21.55 b 31.79 a 31.17 a 23.39 b

38.22 31.30 35.00 38.22

a b ab a

CK 100 200 300

14.74 31.05 22.16 22.15

c a b b

52.80 b 76.76 a 55.99 b 38.96 c

29.82 31.30 33.28 34.51

a a a a

CK 100 200 300

15.98 a 16.97 a 13.01 b 6.59 c

38.46 c 68.36 a 45.55 b 46.87 b

14.74 31.55 41.43 30.06

c b a b

Nandou 032-4

Light

Shade

Table 6 Effect of Si on Chlorophyll pigments of soybean under monocropping and intercropping. Treatments Monocropping

Intercropping

Chl. a

Chl b

carotenoid

Total. chl

a/b

CK

0.060 b

0.016 b

0.011 a

0.077 b

100

0.078 a

0.021 a

0.012 a

0.099 a

200

0.063 b

0.018 b

0.012 a

0.080 b

300

0.061 b

0.017 b

0.011 a

0.079 b

CK

0.061 b

0.017 b

0.010 b

0.079 a

100

0.069 a

0.019 a

0.012 a

0.089 a

200

0.067 ab 0.065 b

0.019 ab 0.018 ab

0.012 a

0.085 a

0.011 ab

0.084 a

3.664 a 3.747 a 3.580 a 3.578 a 3.539 a 3.579 a 3.546 a 3.552 a

300

Means that do not share the same letters in the column differ significantly at p ≤ 0.05.

Means that do not share the same letters in the column differ significantly at p ≤ 0.05.

application of 100 mg/L (Table 6). Under intercropping mode, the chlorophyll a and b contents were significantly improved with the Si application of 100–200 mg/L. However, under monocropping the carotenoid contents were non significant through all treatments of Si but under intercropping 100–200 mg/L of Si improved carotenoid content.

while in the roots it increased significantly. Under normal light and shade conditions, the SSC of Nandou 12 leaves increased significantly after silicon treatment, and reached a maximum value at 100 mg/kg and 200 mg/kg Si. SSC in stems decreased significantly under normal light but increased significantly under 100 mg/kg shade. The SSC in the root system was significantly reduced under both conditions compared with control. Under normal light, the SSC in leaves, stems, and roots of Nandou 032–4 increased with Si application, and was most significant under 200 mg/kg treatment, increasing by 76.2%, 9.9% and 83.9%, respectively. Under shade, Si also increased SSC in leaves, stems, and roots of Nandou 032–4, and the results were most significant with 300 mg/kg Si, resulting in an increase of 94.3%, 72.6% and 40.0%, respec­ tively. The SSC in leaves and stems of shaded Nandou 032–4 increased more significantly than in normal light after Si application, possibly because this variety has a stronger response to silicon under shade in terms of the generation, consumption, and accumulation of soluble sugar. The SSC in Nandou 032–4 was greatest at 300 mg/kg Si. Under all treatments, the SSC of the stems was higher than that of the leaves, which indicates that the stem plays a crucial role in regulating the transport and storage of carbohydrates. As can be seen from Table 4, the starch content in the stems of the two varieties decreased after shading, and increased significantly after applying silicon. Under normal light, the starch content in the leaves of Nandou 12 decreased significantly with the increase of the amount of silicon applied, while the starch content in the roots showed a trend of decreasing first and then increasing. After shading, silicon application significantly reduced the starch content of leaves. Under the CK shade environment, the starch content in the leaves and roots of Nandou 032increased significantly, whereas in stem it decreased. After Si applica­ tion, the starch content of soybean roots increased significantly under normal light, but decreased significantly under shade. Sucrose content in the stems of the two varieties decreased after shading, while sucrose content in normal light and shade increased significantly after Si application (Table 5). Under both light conditions, the sucrose content in Nandou 12 leaves increased first and then decreased with increasing Si concentration. Under normal light, the sucrose content in the leaves of Nandou 032-4 showed the same trend as Nandou 12, which increased first and then decreased. There was no significant difference in sucrose content among all treatments in the roots of Nandou 032–4 under light condition.

3.1.8.3. Yield and yield composition. As shown in Table 7, the number of available branches, the number of available pods, the number of seeds per plant, and the weight of seeds per plant all increased significantly in the net cropping environment. The values of effective branching, effective pod number, and seed weight per plant were highest with 100 mg/kg of Si treatment. The number of ineffective pods increased at 100 mg/kg, but decreased at 200 mg/kg and 300 mg/kg of Si treatment. The 100-seed weight was reduced by 5.0% at 100 mg/kg of Si treatment. The yield of mono-cultured soybeans increased significantly with Si appli­ cation: 200 mg/kg > 100 mg/kg > 300 mg/kg. The effective pod number, seed number per plant and seed weight per plant also increased significantly after Si application under intercropping (shading), thereby increasing the yield. Si also increased the number of branches, and the difference was significant at 300 mg/kg. Application of 200 mg/kg of Si increased the yield under mono-cropping by 17% and under intercrop­ ping by 24%. These results suggest that foliar application of Si under intercropping could prevent the negative effects of shading. 3.1.8.4. Relationship between yield and silicon content. Correlation analysis results showed (Fig. 3) that soybean yield was significantly positively correlated with the accumulated of silicon content in plants (r = 0.885**). Thus, the accumulation of silicon in the plant was increased after intercropping with soybean, thus improving soybean yield. 4. Discussion Si has been well known to regulate endogenous phytohormones (GA and JA), and expression of essential proteins. In present study, higher concentration of silicon significantly reduced plant height and increased stem diameter. Recently, previous studies reported that exogenous Si application significantly increased physiologically active endogenous gibberellin (GA) (Jang et al., 2018; Khan et al., 2020), which might be the reason of reduced plant height under higher concentration of Si application. Better plant growth is the result of more efficient nutrient uptake. Previous studies reported that increases in biomass and dry weight of plants had a positive linear correlation with nutrient uptake and photosynthetic rate (Guo et al., 2019). In the present study, Si

3.1.8.2. Effect of Si on photosynthetic pigments. Under monocropping the chlorophyll a and b contents were significantly improved with the Si 49

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Table 7 Effect of Si on yield and yield composition of soybeans under mono-cropping and intercropping. Treatment Monocropping Intercropping

CK 100 200 300 CK 100 200 300

Effective branch

Effective pods per plant

Ineffective pods per plant

Beans per plant

Bean weight per plant(g)

Weight of 100 seeds (g)

Yield (kg/ hm2)

Yield increase rate (%)

5.33 c 9.67 a 8.33 b 9.33 ab 4.33 b 5.00 b 5.67 b 8.00 a

83.67 b 168.33 a 154.33 a 168.00 a 54.33 c 71.33 b 116.67 a 111.33 a

7.67 ab 10.33 a 6.33 b 5.33 b 5.67 a 5.67 a 7.00 a 5.67 a

102.00 c 151.33 b 181.67 a 155.00 b 81.67 d 99.67 c 145.33 a 128.33 b

25.85 44.40 41.18 42.13 15.33 16.91 17.00 15.00

17.27 16.41 17.60 18.62 18.09 16.40 17.87 17.66

2485.00 d 2840.33 b 2917.67 a 2813.00 c 1333.33 d 1591.33 b 1660.00 a 1469.67 c

– 14.30 17.41 13.20 – 19.35 24.50 10.23

b a a a b a a b

b c b a a c ab b

Means that do not share the same letters in the column differ significantly at p < 0.05.

2017). In the current study, Si may regulate new adventitious root for­ mation and primary root biomass accumulation by nitric oxide and reactive oxygen species signaling. Changes in chlorophyll pigments in intercropping significantly affected photosynthetic efficiency. Under intercropping, shade results in lower chlorophyll content and reduced photosynthetic rate (Fan et al., 2018). Si application is considered a potential treatment to impart resistance to abiotic stresses on the photosynthetic machinery (Tripathi et al., 2016a). In the present study, we observed that shade significantly reduced the chlorophyll content of soybean plants, and Si application promoted chlorophyll biosynthesis under intercropping and mono­ cropping (Table 6). In support of our current findings, recent studies have shown that Si application increased the content of chlorophyll and non-structural carbohydrates, and up-regulated genes related to light harvesting complex II, which increased the photosynthetic rate (Teixeira et al., 2020; ZHANG et al., 2018). Furthermore, it has been reported that photosynthetic capacities and efficicency of PSII of crops’ applied Si are improved by the enlarged size of chloroplasts and the increased number of grana in leaves (Xie et al., 2014). Increase in Gs were noticed after Si exposure under normal light and shade conditions. The Si application of 100–200 mg/kg significantly improved Gs of both cultivars under normal light and shade conditions. Similar reports on strawberry (Fragaria chiloensis (L.) Mill.) (Wang et al., 2009), tomato (Lycopersicon esculentum Mill.), rice (Oryza sativa L.) (Cao et al., 2013; Gao et al., 2011), and wheat (Triticum aestivum L.)

Fig. 3. The relationship between silicon content and yield of soybean.

application increased the root length and root surface area of soybeans under intercropping and monoculture, and the increased growth might be due to increased nutrient uptake. Increased biomass accumulation has been linked to increased nutrient uptake because of improved root growth of soybean plants and may also be associated with enhanced production of nitric oxide and ROS signaling (Kushwaha et al., 2019). Similar to our results, a past study reported that Si application increased uptake of phosphorus and nitrogen (Greger et al., 2018; Kostic et al.,

Fig. 4. Model depicting the Si mediated alleviation of shading stress in soybean. 50

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Plant Physiology and Biochemistry 159 (2021) 43–52

showed that Gs can be increased by Si fertilizer. Increases in Gs, which regulates gas exchange (CO2 and water), can allow plants under well-watered growth conditions to increase their CO2 uptake and sub­ sequently enhance photosynthesis (Kusumi et al., 2012). Under normal water conditions, the values Gs of increase together with the increasing of photosynthetic rate, by which crops regulate stomatal conductance to reduce water loss. Shade has negative effects on stem strength and lignin content of soybean (Hussain et al., 2019d). Previous studies reported that under shade, Si affects the composition of plant cell walls by altering linkages of non-cellulosic polymers and lignin biosynthesis enzymes (Hussain et al., 2020c). In our findings, shade stress reduced stem diameter, stem strength and lodging resistance, but this was reversed by application of Si. The reason for improved lodging resistance after Si application might be the regulation of lignin biosynthesis enzymes and genes (Hussain et al., 2020c). Si plays a dynamic role in plant growth and development, and one of its main actions is to offset the negative effects of abiotic stress (Kim et al., 2012, 2017). Studies have shown that different concentrations of Si caused different effects. Improvements in soybean yield due to the use of Si was a result of increase in the number of pods per plant, the number of seeds in the pod, and the weight of 100 seeds (Artyszak, 2018). In another field experiment conducted in Poland, Si increased the number of pods in soybeans by 18%, and the average seed yield per plant by 21% (Kornd¨ orfer and Lepsch, 2001). Studies conducted in China in 2005–2006 on application of Si to the soil recorded an increase in soy­ bean yield of 7.5–13.6% (Liang et al., 2015b; Wang et al., 2001). In our findings, Si application resulted in a 19% increase in yield.

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5. Conclusions The Foliar application of Si can be used effectively for soybean in normal light and shade conditions. The study exhibits that Si application was closely related to the values of different parameters of chlorophyll contents (Chl a, Chl b and Carotinoid), lodging resistance, photosyn­ thetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and root architecture of soybean plants under normal light and shade con­ ditions. In this study the results showed that in normal light and shade conditions, the optimal dose of Si application was 100–200 mg/kg, under which photosynthetic efficiency and yield of soybean plants were significantly increased. Thus, Si can be used in intercropping system as a spray fertilizer for the purpose to alleviate the stress effects of shading through regulating morphological characteristics, photosynthetic ca­ pacity and carbohydrate synthesis of leaves, stems and roots, hence enhancing yield and resistance (Fig. 4). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was funded by the National Key Research and Develop­ ment Program of China (2018YFD1000905) and Project APVV-18-0465. Dr. Sajad Hussain is thankful to Mr. Ijaz Yaseen for his technical assis­ tance during experiment. References Abbas, T., Balal, R.M., Shahid, M.A., Pervez, M.A., Ayyub, C.M., Aqueel, M.A., et al., 2015. Silicon-induced alleviation of NaCl toxicity in okra ( Abelmoschus esculentus ) is associated with enhanced photosynthesis, osmoprotectants and antioxidant metabolism. Acta Physiologiae Plantarum 37, 6.

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