MWCNTs Alleviated saline-alkali stress by optimizing photosynthesis and sucrose metabolism in rice seedling

ABSTRACT

Saline and alkali stress affects the growth and development, survival rate, and final yield of rice, while new nano materials can have a positive effect on rice growth. In order to investing the effects of carboxymethyl multi walled carbon nanotubes (MWCNTs) on the growth and development of rice seedlings under salt alkali stress, rice seedlings were cultured using rice variety “Songjing 3” using nutrient solution water culture method. The effects of MWCNTs on water absorption capacity, leaf photosynthesis, and sucrose metabolism of rice seedlings under 50 mmol/L saline-alkali stress (1NaCl: 9Na₂SO₄: 9NaHCO₃: 1Na₂CO₃) conditions were investigated. The results showed that MWCNTs can improve the water use ability of roots and leaves, especially the water absorption ability of roots, which provides a guarantee for the improvement of rice biomass and the enhancement of leaf photosynthetic capacity under adverse conditions. After treatment with MWCNTs, the photosynthetic rate (P_n), stomatal conductance (g_s), and transpiration rate (T_r) of leaves increased significantly, and the photochemical quenching value (qP), photochemical quantum efficiency value (F_v/F_m), and electron transfer rate value (ETR) of chlorophyll fluorescence parameters increased significantly, which is beneficial to the improvement of the PSII photosynthetic system. MWCNTs treatment promoted the increase of photosynthetic pigment content in leaves under salt and alkali stress, improved the ratio of Chla and Chlb parameters, increased the activities of key photosynthetic enzymes (RUBPCase and PEPCase) in leaves, increased the value of total lutein cycle pool (VAZ), and significantly enhanced the deepoxidation effect of lutein cycle (DEPS), which can effectively alleviate the stomatal and non stomatal constraints on leaf photosynthesis caused by salt and alkali stress. MWCNTs treatment significantly enhanced the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) under salt and alkali stress, and decreased the activities of soluble acid invertase (SAInv) and alkaline/neutral invertase (A/N-Inv), indicating that MWCNTs promoted sucrose synthesis while inhibiting sucrose decomposition, thereby promoting sucrose accumulation in rice leaves. This study can provide theoretical and experimental basis for the application of MWCNTs to the production of rice under salt and alkali stress, and can find a new way for rice production in saline and alkaline lands.

KEYWORDS:

• Rice
• MWCNTs
• saline-alkali stress
• photosynthesis
• sucrose metabolism

1. Introduction

Under the background of the past era of the current COVID-19, as well as the current background of frequent natural disasters and gradual reduction of agricultural land, it is an important means to ensure the effective supply of rice production by improving the adaptability of crops to adversity, developing and utilizing saline alkali land, and effectively using degraded agricultural land. Applying new technologies and methods such as plant nanobiology to improve rice stress resistance to ensure food production is a feasible alternative. During the growth and development of crops, they will be affected by a variety of abiotic stress, and soil salinization is one of the common stress in agricultural production.^1,2 Currently, soil salinization is becoming increasingly serious, with the area of saline and alkaline land in Asia being approximately 2.9 × 10⁸ hm². There are about 33.33 million hm2 of saline soil in China, accounting for about 10% of the cultivated land area³. Rice is an important food crop, with nearly 1/2 of the world’s population relying on rice as their staple food. In China, about 2/3 of the population rely on rice as their staple food, and the rice planting area accounts for about 27% of the grain planting area. Soil salinization affects China’s rice planting and also threatens food security. Saline and alkali stress can lead to plant height shortening, leaf area reduction, and tillering reduction, thereby reducing photosynthetic efficiency.^3,4 Traditional genetic breeding, water and fertilizer operations, and field management measures have shortcomings. Therefore, plant nanotechnology emerged as the times require. The strategy for traditional agriculture to control pests and diseases and improve agricultural production efficiency is mainly to apply chemical fertilizers and pesticides. In the long run, in addition to reducing soil fertility, it will also cause serious environmental pollution, cause great pressure on the ecosystem, and also affect human health. In many fields, including agricultural production, nanotechnology has the potential to solve related problems caused by traditional agricultural production by changing existing technologies, such as environmental pollution caused by the use of chemical pesticides for pest control, and the low efficiency of improving and developing new high-yield and stress resistant crop varieties.⁵ As an emerging frontier crossing field, it originates from the deep integration of nanotechnology and agricultural science. Due to its small size, nanomaterials can be absorbed by cells through endocytosis after penetrating plant cell walls, thereby affecting plant growth.⁶

Using nano materials to treat seeds can enhance the activity of the seeds, improve the activity of various enzymes in the seeds, thereby promoting the growth of plant roots, improving the absorption of water and fertilizer by plants, promoting metabolism, and further improving the plant’s insect resistance, disease resistance, and various stress resistance abilities on the basis of the agronomic characteristics of the original varieties, achieving the effects of increasing production and improving quality. Plant nanobiology technology is a newly emerging frontier interdisciplinary field in the world, with great application potential in the fields of plant stress resistance, plant transgenic, and plant nanobionics. This technology can be used to improve the ability of crops to resist salt, drought, high and low temperatures, disease and insect pests.^7–11 Nanomaterials have a small volume (1–100 nm), good biocompatibility, and special surface effects and optical properties. They have made considerable progress in nano pesticides, nano carriers, and nano plant stress resistance.^10–12 An important mechanism for plant roots to absorb nutrients is to dissolve and absorb substances from the root zone environment through organic acids secreted by plant roots. Nanometer mineral particles are easily dissolved and absorbed by this weak acid due to their large specific surface area, large surface bonds and charges, and high chemical activity. This is the mechanism by which plants absorb and utilize nanoscale insoluble mineral particles. In improving crop stress resistance and efficient agricultural production in the future, nanotechnology has great potential for application and can provide green and efficient solutions for agricultural production in the future.^11–15

Carbon nanotubes (CNTs) are currently widely used as new engineering nanomaterials. Generally, CNTs can be divided into single walled carbon nanotubes (SWCNTs) and multi walled carbon nanotubes (MWCNTs) based on the number of graphene sheets they contain.¹⁶ The germination rate of tomato seeds treated in medium supplemented with MWCNTs increased by 40% compared to the control because MWCNTs infiltrated into the seed coat to promote seed water absorption and thereby stimulate seed germination.¹⁷ Low concentrations of MWCNTs (20 mg/L) promote the germination of corn seeds, and the accumulation of elements such as Ca and Fe during the germination process has been observed.¹⁸ Different crop seeds exhibit different sensitivities to the addition of different concentrations of SWCNHs.¹⁹ The application of CuO NPs has significantly promoted the growth and grain yield of maize.²⁰ After applying TiO2 NPs to mung beans, the activities of phosphatase and phytase in the root soil significantly increased.²¹ Under low concentration conditions, ZnO NPs can significantly promote the increase of rhizome length of asparagus mosaic seeds, while 100 mg/L ZnO NPs has a significant inhibitory effect on plant rhizome growth.²² Carbon nanotubes can enter plant tissue cells, thereby affecting the growth and development of plants, and affecting material metabolism in plants.¹⁷ Studies have found that single-walled carbon nanotubes have a promoting effect on the elongation of onion and cucumber roots.²³ Low concentration multi walled carbon nanotubes can penetrate the seed coat of tomato seeds, improving the water absorption of the seeds, thereby promoting seed germination and root and stem growth.¹⁷ For example, MWCNTs can significantly increase the germination rate and root extension of radish, rapeseed, ryegrass, lettuce, corn, and cucumber seeds.^17,24 The photochemical effects of some nanomaterials such as TiO2 can produce superoxide compounds of reactive oxygen species, increasing the resistance of seeds and promoting their rapid uptake of water and oxygen. Nanomaterials penetrate into the seeds, increasing their uptake of water, thereby promoting the germination rate of the seeds.²⁵ In addition to promoting tomato growth and increasing its biomass, multi-walled carbon nanotubes can also affect the composition of microorganisms in the soil, increasing the number of thick-walled bacteria and bacteroides.²⁶ Some studies have also found that low concentrations of multi walled nanotubes and oxidized multi walled nanotubes can increase the water content of mustard seeds, improve the water absorption and transportation efficiency of root tissue.²⁷

Currently, research on carbon nanotubes is mainly focused on new energy, new materials, and biosensors.^28–30 The research on carbon nanotubes in agriculture is relatively small, mainly focusing on the regulation of plant growth and development, plant disease prevention, environmental protection, and other aspects.^31–33 Carbon nanotubes are the most promising nanomaterials. However, up to now, most studies have focused on the interaction between carbon nanotubes and mammalian cells. Carbon nanotubes can easily penetrate animal cell membranes and exhibit extremely low cytotoxicity. Due to the thicker cell walls of plants, there are few studies on the effects of nanomaterials on plants^34,35 Although carbon nanotubes can affect the germination of plant seeds and the development of root systems, their effects vary due to the types of nanomaterials and plant genotypes, and the mechanism of these effects is currently unclear. Existing research has found that carbon nanotubes affect the expression of genes such as aquaporins, thereby affecting the absorption and transportation of water by plants. Therefore, they have broad application prospects in agriculture, and some achievements have been achieved. However, it is necessary to further study whether carbon nanotubes can improve the salt and alkali tolerance characteristics of rice. To achieve nano agriculture, it is necessary to conduct research on the effects of carbon nanotubes on the growth of rice seedlings. By exploring the effects of carbon nanotubes on the water status of rice plants, leaf photosynthesis, and sucrose metabolism under salt and alkali stress, it is necessary to clarify the promotion mechanism of carbon nanotubes on the growth of rice seedlings, providing a theoretical basis for further research on the possible yield increase mechanism of carbon nanotubes on rice.

2. Materials and methods

2.1. Experimental materials

Rice variety “Songjing 3” was chosen as experimental material, and the seed was the first erect panicle type variety in cold region of Heilongjiang Province. The plant type was convergent, the panicle and leaf were erect, the stem was strong and resistant to lodging, and suitable for close planting. It was bred by Wuchang Rice Research Institute of Heilongjiang Academy of Agricultural Sciences. The experiment was completed jointly by Wuchang Rice Research Institute of Heilongjiang Academy of Agricultural Sciences and the Laboratory of Agricultural College of Northeast Agricultural University. Carboxylated multi-walled carbon nanotubes (MWCNTs) were purchased from Zhongke Shidai Nano Chengdu Organic Chemistry Co., Ltd., and the specific parameters are as follows: OD (8–15 nm), Length (0.5–2 μm), Purity>95%, Special surface area (233 m²/g), Electric conductivity>100 s/m. We weighed 20 mg dry powder of multi walled carbon nanotubes and suspended it in 1000 ml of ultrapure water. Then, they were ultrasonically treated in the ultrasonic cleaner at 320 w for 30 min to obtain multi walled carbon nanotube suspensions with concentrations of 20 mg/L respectively. Ultrapure water was used as a blank control test. Based on the preliminary experimental results and previous references, we set the concentration of typical saline-alkali stress as 50 mmol/L Saline alkali stress (1 NaCl: 9 Na₂SO₄: 9 NaHCO₃: 1 Na₂CO₃).^36,37

2.2. Experimental design

We selected the seeds with plump granules and consistent size, disinfected them with 3% sodium hypochlorite solution for 15 minutes, washed them with ultrapure water, soaked them in warm water at about 30°C for 36 hours, and then transferred them to an artificial climate incubator at 26°C for germination. After conventional seed germination, we screened seedlings with 5 mm long buds and suspended them in Hoagland nutrient solution for rice.^36,38,39 The Hoagland nutrient solution culture method above is adopted. 100 rice plants are suspended in each tank and treated when the seedlings grow to 3 leaves and 1 heart. The seedling growth test was divided into four treatments: (1) clear water (CK); (2) 20 mg/L Carboxylated multi-walled carbon nanotubes (MWCNTs); (3) 50 mmol/L Saline-Alkali stress (SA); (4) 20 mg/L MWCNTs +50 mmol/L Saline Alkali stress (MWCNTs+SA), each treatment was repeated three times. The culture conditions are as follows: the photoperiod is 12/12 (day/night), the temperature is (28 ± 1)℃/(25 ± 1)℃ day/night, and the light intensity is 400 μmol/m²·s, and the relative humidity is 60%-70%. The solution of each treatment group was changed every 2 days. At 0 h, 24 h, 48 h, 72 h, 96 h and 120 h after seedling treatment, an appropriate amount of allelic leaves and roots were taken for the determination of various physiological and biochemical indicators. All tests were repeated for three times.

2.3. Measurement and methods

2.3.1. Determination of leaf relative water content (RWC)

Take rice leaves that have been treated for 0, 24, 48, 72, 96, and 120 hours, weigh their fresh weight (mf), and record them. Soak the leaves in distilled water overnight, absorb surface moisture, and weigh their saturated weight (mt). Place the saturated leaves in an oven at 105°C for 15 minutes, dry them to constant weight at 75°C, and weigh their dry weight (md). Leaf relative water content is obtained by the following calculation equation: w(RWC)=(mf−md)/(mt−md)×100%.⁴⁰

2.3.2. Determination of leaf water potentials (Ψ_w)

Take rice leaf samples from 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h after treatment, cut them up, and place them in a special sample box for WP4C water potential meter (diameter 40 mm ×height 12 mm). We use a dew point water potential meter (WP4C Dewpoint Potential Meter, Decagon Devices Inc., Pullman, WA, USA) to measure the water potential of the leaves, and record the water potential data after the reading is stable.⁴¹

2.3.3. Determination of root hydraulic conductance (L_p)

The measurement was completed by using plant water potential pressure chamber instrument (Type 3115, Beijing). L_p is calculated according to the following equation. L_p=J_v/P, Where J_v (m/s) is the flow rate; P is the externally applied pressure (MPa).^42,43

2.3.4. Determination of Chlorophyll a (Chla), Chlorophyll b (Chlb) and Carotene (Car) contents

We weigh 0.5 g of the sample, grind 5 ml of acetone (80% v/v), centrifuge 10,000 g, measure the absorbance with a spectrophotometer (UV-5500) at 470, 645 and 663 nm, and calculate Chla and Chlb according to Arnon’s equation. Car = 8.73 *OD₄₇₀ +2.11*OD₆₆₃-9.06*OD_645.^44,45

2.3.5. Determination of xanthophyll cycle components (A,V,Z) and ratio

We used Shimadzu LC-20A high-performance liquid chromatography to analyze the contents of Zeaxanthin (Z), Antheraxanthin (A) and Violaxanthin (V) in xanthophyll circulation of fresh rice leaves. Grind 0.5 g of the sample with liquid nitrogen, extract lutein pigments with acetone for 1 min, extract at 2500 g, and centrifuge for 10 min. The supernatant obtained after centrifugation needs to undergo 0.2 μm filter filtration. We immediately took 25 µL of the filtrate for sample analysis and used a Spherisorb C₁₈ column (4.6 × 250 mm, 5 μm Kromasil). Elute with solution A for 10 min, linear gradient for 4 min and elute with solution B for 30 min. The deepoxidation state of the lutein cycle (DEPS) was calculated using he calculation equation (Z+A)/(V+A+Z).^46,47

2.3.6. Determination of gas exchange parameters (P_n, g_s, T_r, WUE, L_s, C_i)

We use a portable photosynthetic system tester LI-6400 (LI-CORLink, USA) to measure net photosynthetic rate (P_n)、stomatal conductance (g_s)、transpiration rate (T_r)、water use efficiency (WUE)、Stomatal limitation (L_s) and intercellular carbon dioxide concentration (C_i) in rice functional leaves. After treatment of rice leaves, analysis and determination were conducted at 0, 24, 48, 72, 96, and 120 hours, while functional leaves were selected for determination. In order to reduce the impact of external environmental fluctuations on the measurement results, all gas exchange parameters in this test were measured from 9:00 to 17:00, and were completed in an environmentally controllable growth box. The environmental control in the growth chamber is: temperature 28°C, humidity 60%, and light intensity 500 µmol/m².s. Before the measurement of leaf gas exchange parameters begins, the rice plant adapts in the growth box for at least 1 hour in advance. During measurement, rice leaves are sandwiched between the leaf chambers of the photosynthetic apparatus, with the light intensity set at 1500 µmol/m².s, the CO₂ concentration controlled at 400 µmol/mol, the leaf temperature at 28°C, and the relative humidity at 60%. During measurement, insert the blade for 3.5 min and record the data of gas exchange parameters. Repeat the measurement for six times for each process.^48,49

2.3.7. Determination of chlorophyll fluorescence parameters (F_m, F_v/F_m, ФPSII, ETR, qP, NPQ)

Chlorophyll fluorescence was measured using a PAM-2100 chlorophyll fluorescence meter (Walz, Germany). After treatment of rice leaves, analysis and determination were conducted at 0, 24, 48, 72, 96, and 120 hours, while functional leaves were selected for determination. In order to reduce the impact of external environmental fluctuations on the measurement results, all gas exchange parameters in this test were measured from 9:00 to 17:00, and were completed in an environmentally controllable growth chamber. After 30 minutes of dark adaptation, the measured light (<<0.05 μM m⁻² s⁻¹) obtains minimum fluorescence (F_o) and passes through saturated pulsed light (0.8s; 8000 μM m⁻² s⁻¹) to obtain maximum fluorescence (F_m). By turning on the active light (300 μM m⁻² s⁻¹) Measure photosynthetic steady-state fluorescence (F_s) and turn on saturated pulsed light (8000 μM m⁻² s⁻¹) to obtain the maximum fluorescence (F_m’), turn off the action light, immediately turn on the far red light, and obtain the minimum fluorescence (F_o’) under the light. PSII maximum photochemical efficiency F_v/F_m =（F_m-F_o）/F_m; Photochemical quantum efficiency of optical systems IIΦPSII=（F_m’ - F_s）/F_m’; Electron transfer rate: ETR = (ФPSII × 0.5 ×PPFD × 0.84), where PPFD is the luminous flux density; The photochemical quenching coefficient qP =（F_m’-F_s）/（F_m’-F_o’）; And non photochemical quenching coefficient NPQ =（F_m-F_m’）/F_m’.⁵⁰

2.3.8. Determination of RUBPCase (ribulose 1,5-diphosphate carboxylase/oxygenase) and PEPCase (phosphoenolpyruvate carboxylase) in leaves

After treatment of rice leaves, analysis and determination were conducted at 0, 24, 48, 72, 96, and 120 hours, while functional leaves were selected for determination. Take 0.5 g of fresh leaf sample, add a small amount of quartz sand and 3 mL of precooled extraction liquid for grinding extraction. The extract contains 100 mmol/L Tris-HCl (pH 7.8), 10 mmol/L MgCl₂, 1 mmol/L EDTA, and 20 mmol/L β- mercaptoethanol, 10% (W/V) glycerin, and 1% PVP. The filtered filtrate was centrifuged at 4°C at 15,000 rpm for 10 minutes, and then the supernatant was taken for enzyme activity measurement.

The reaction system for the determination of RuBPCase active enzyme was 3.2 mL, and the reaction mixture consisted of 100 mmol/L Tris-HCl buffer (pH 8.0), 100 mmol/L MgCl₂, 50 mmol/L adenine nucleoside triphosphate (ATP), 50 mmol/L dithiothreitol (DTT), 2.0 mmol/L NADH, 1.0 mmol/L EDTA-Na₂, 200 µ mol/L NaHCO₃, 0.1 mL distilled water, 3-phosphate glycerate kinase (PGK)/3-phosphate glyceraldehyde dehydrogenase (GAP-GDH), 0.1 mL enzyme extract. Maintain a constant temperature water bath at 30°C for 10 minutes, measure the absorbance value E0 at 340 nm, and finally add 0.1 ML 9.0 mmol/L 1,5-bisphosphate to start the reaction. Immediately measure the change in light absorption at intervals of 10 s to 15 s.⁵¹

The reaction system for the determination of PEPCase active enzyme is 1 mL, and the reaction mixture contains 50 mmol/L Tris-HCl (pH 7.8), 10 mmol/L MgCl₂, 0.25 mmol/L EDTA, 5.0 mmol/L NaHCO₃, 2.0 mmol/L DTT, 0.1 mmol/L NADH, 4 units MDH, 2.0 mmol/L PEP. The reaction is started by adding enzyme solution, and the decrease in optical density at 340 nm wavelength is tracked on a Varian 100 spectrophotometer and the measurement time is 3 min.⁵²

2.3.9. Determination of Sucrose Synthetase (SS) and Sucrose Phosphate Synthase (SPS)

Take 0.5 g of blade tissue, grind it with liquid nitrogen, transfer the ground powder to a centrifuge tube, and add 3 ml of Hepes-NaOH buffer solution with pH 7.5; Place them in a refrigerator at 4°C for 30 minutes. After removal, freeze and centrifuge at 12,000 g and 4°C for 10 minutes. Take the supernatant and add (NH4)₂SO₄ to saturate the solution to 80%. Repeat placing in the refrigerator and freeze centrifuging once. Discard the supernatant, add 1 ml of Hepes-NaOH buffer solution with pH 7.5 to the precipitation, and fully dissolve the precipitation to obtain the enzyme solution. The reaction system for SPS activity determination includes enzyme solution 50 µl, Hepes-NaOH buffer 50 µl, 50 mM MgCL₂ 20 µl, 150 M UDPG 20 µl, and 50 mM fructose 6-phosphate 20 µl. Mix well and place in a 30°C water bath for reaction for 30 min, then add 200 µl of 40% NaOH solution and place in a boiling water bath for 10 min. Boiled enzyme solution was used as blank control. The difference between SS activity measurement and SPS activity measurement is that fructose is used instead of fructose 6-phosphate, and the activity of SS enzyme can be measured by performing other steps according to the above method.^53,54

2.3.10. Determination of invertase enzyme (SAInv) and Alkalinity/Neutral Enzyme (A/N-Inv)

Weigh 0.5 g of blade tissue, grind with liquid nitrogen, transfer the ground powder to a precooled centrifugal tube, and add 4 mL of extraction buffer solution with pH 7.5. The buffer contains 150 mmol/L potassium phosphate buffer, 0.1% β- mercaptoethanol, 5 mmol.L MgCl₂, 0.05% BSA, and 0.05% Triton X100 were placed in a refrigerator at 4°C for 20 minutes, and centrifuged at 4°C and 12,000 g for 30 minutes. Transfer the supernatant, add (NH4)₂SO₄ to the supernatant, and continuously shake it until the saturation of the final solution reaches 80%. Place it in a refrigerator at 4°C for 30 minutes, centrifuge at 4°C for 12,000 g for 20 minutes. Discard the supernatant, and add 1 mL of demineralized buffer solution with pH 7.5 to the sediment The buffer consists of 15 mmol/L potassium phosphate buffer, 0.01% β-sulfhydryl ethanol, 0.25 mmol/L MgCl₂, and 0.05% BSA are composed, and the protein precipitate is completely dissolved by sufficient shaking to obtain the enzyme solution. The reaction system for determining the activity of SAInv consists of 0.7 ml of 80 mmol/L acetic acid sodium acetate buffer (pH 4.5), 0.2 mL of 100 mmol/L sucrose, and 0.1 mL of enzyme extract. We mix well and place in a 37°C water bath for 30 minutes before taking out. Add 1.5 mL of DNS reagent. After fully mixing, place each test tube in a boiling water bath and boil for 5 minutes. After cooling to room temperature, measure its absorbance at 540 nm. The reaction system for the determination of A/N-Inv activity is basically the same as that for SAInv, except that a potassium phosphate buffer with pH = 7.5 is used instead of a acetic acid sodium acetate buffer with pH = 4.5.^53,55

2.4. Statistical analysis

Use Excel 2013 to organize and calculate data. Statistical software (Tallahassee, FL, USA) was used for variance analysis, and the least significant difference (LSD) method was used to compare the difference significance of the average value at the 0.05 level.

3. Results

3.1. Leaf Relative Water Content(RWC)、Leaf water potentials(Ψ_w) and root hydraulic Conductance(L_p)

Table 1 shows that the values of Ψ_w and RWC of rice seedling leaves showed a downward trend after 24 h to 120 h of saline alkali stress treatment, while MWCNTs+SA treatment could improve the water balance of rice seedlings under saline alkali stress. Taking 120 h after stress as an example, compared with CK, the RWC and Ψ_w decreased by 27.88% and 121.89% respectively. Compared with SA treatment, MWCNTs+SA treatment can alleviate the decreasing range of RWC and Ψ_w, ensuring the water metabolism balance of rice will not be broken rapidly under adverse conditions, and the normal physiological and biochemical processes could be maintained. Taking 120 h after stress as an example, compared with SA treatment, Ψ_w increased by 4.96%% and RWC increased by 13.95% under MWCNTs+SA treatment. As shown in Table 1, the L_p value of rice roots under saline alkali stress decreased significantly. Compared with CK, the L_p after SA treatment decreased by 14.41%, 30.35%, 23.47%, 30.2% and 32.91% at 24 h, 48 h, 72 h, 96 h and 120 h, respectively, which will directly inhibit the water absorption of rice roots and affect the growth and development of rice. However, the application of MWCNTs could significantly alleviate the decrease of root L_p value of rice seedlings under saline alkali stress. Taking 24 h, 72 h and 120 h after stress as examples, compared with SA treatment, root L_p under MWCNTs+SA treatment increased by 10.53%, 16% and 21.14% respectively. The above results indicate that the addition of MWCNTs to the exogenous application can improve the water use capacity of rice roots and leaves, especially the improvement of root water absorption, which lays a foundation for the improvement of dry matter accumulation in the later stage of rice under saline alkali stress (Table 1).

Table 1. Effects of MWCNTs on leaf RWC, leaf Ψ_w and root L_p under saline-alkali stress in rice.

Treatments	Index	0h	24h	48h	72h	96h	120h
CK	Leaf relative water content(RWC, %)	83.42±0.97^a	83.97±1.05^a	82.08±1.05^a	81.59±1.15^a	80.43±1.05^a	77.99±0.76^a
MWCNTs		84.15±1.05^a	83.16±1.15^a	81.54±0.93^a	80.59±1.05^a	81.15±0.95^a	79.74±0.72^a
SA		82.08±1.15^a	74.07±1.05^b	69.48±0.93^c	63.27±1.15^c	56.88±0.98^c	56.25±0.55^c
MWCNTs+SA		82.17±1.42^a	76.86±0.95^ab	72.27±0.84^b	67.14±1.05^b	64.98±1.05^b	64.10±0.58^b
CK	Leaf water potentials(Ψw,Mpa)	−0.679±0.04^a	−0.670±0.02^a	−0.688±0.02^a	−0.707±0.02^a	−0.725±0.03^a	−0.763±0.01^a
MWCNTs		−0.688±0.03^a	−0.670±0.03^a	−0.698±0.02^a	−0.670±0.03^a	−0.619±0.03^a	−0.605±0.02^a
SA		−0.688±0.02^a	−1.228±0.01^c	−1.414±0.03^c	−1.572±0.02^c	−1.614±0.04^c	−1.693±0.02^c
MWCNTs+SA		−0.698±0.03^a	−0.911±0.02^b	−1.144±0.02^b	−1.358±0.02^b	−1.507±0.02^b	−1.609±0.03^b
CK	Root hydraulic conductance(Lp)	10.80±0.20^a	11.10±0.18^a	11.20±0.18^a	9.80±0.18^a	9.60±0.19^a	9.45±0.13^a
MWCNTs		11.20±0.19^a	11.50±0.19^a	12.40±0.18^a	10.80±0.19^a	10.40±0.19^a	9.88±0.15^a
SA		10.90±0.18^a	9.50±0.17^b	7.80±0.19^c	7.50±0.18^c	6.70±0.20^c	6.34±0.12^c
MWCNTs+SA		11.30±0.19^a	10.50±0.18^ab	9.20±0.18^b	8.70±0.18^b	7.90±0.18^b	7.68±0.15^b

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

3.2. Contents of Chla, Chlb, car and ratio of Chla/Chlb, car/Chl(a+b)

Photosynthesis is the most basic physiological process of plants. Various natural factors, such as drought, flooding, low temperature, salt damage, etc., will directly or indirectly affect photosynthesis. The structure of chloroplasts of plant leaves is often destroyed, the synthesis ability of photosynthetic pigments is reduced, and the content of chlorophyll and carotenoids is reduced, thus affecting the smooth progress of the primary reaction under photosynthesis.⁵⁶ Chla and Chlb are the main pigments that absorb and transfer light energy, which can improve the light capture efficiency. The ratio of Chla/Chlb is closely related to the stability of PSII light capture complex. Table 2 shows that MWCNTs treatment can increase the content of Chla, Chlb and Chl(a+b) in rice leaves. Compared with CK, the content of Chla, Chlb, Chl(a+b) and the ratio of Chla/Chlb in leaves after 120 hours of saline alkali stress treatment showed a significant downward trend at first, and decreased by 43.38%, 30.65%, 40.98% and 18.36% respectively. Compared with SA treatment, MWCNTs+SA treatment significantly increased the content of photosynthetic pigments in rice leaves. After MWCNTs+SA treatment, the content of Chla, Chlb, Chl (a+b) and the ratio of Chla/Chlb in rice leaves increased by 15.93%, 12.9%, 15.26% and 2.67%, respectively. This shows that MWCNTs can promote the leaf chlorophyll synthesis ability under salt and alkali stress, slow down the process of chlorophyll decomposition and transformation, and thus enhance the photosynthesis of rice leaves under salt and alkali stress. Car exist in chloroplasts of plants. Carotenoids are important components of thylakoid membranes, which can quench the dissipated energy in various forms such as excited state or lutein cycle. The results showed that the car content in rice leaves under saline alkali stress decreased, while the Car/Chl (a+b) ratio increased significantly. For example, the Car content and Car/Chl (a+b) ratios in SA treated leaves were 0.242 and 0.113, respectively, lower than 0.301 and 0.083 in the control. MWCNTs+SA treatment can slow down the decline of carotenoid content in leaves under saline alkali stress, and increase the ratio of Car/chl (a+b) under Cd treatment. For example, compared with CK, the Car content and Car/Chl(a+b) ratio after MWCNTs treatment increased by 53.16% and 18.07% respectively. Compared with SA treatment, the car content and Car/Chl (a+b) ratio of MWCNTs+SA treatment increased by 21.9% and 6.19% respectively (Table 2).

Table 2. Effect of MWCNTs on different photosynthetic pigment contents and ratio under saline-alkali stress in rice (120 h).

Treatments	Chla	Chlb	Chl(a+b)	Chla/Chlb	Car	Car/Chl(a+b)
CK	2.939±0.042^b	0.682±0.035^b	3.621±0.068^b	4.309±0.085^a	0.301±0.017^b	0.083±0.004^b
MWCNTs	3.885±0.033^a	0.822±0.034^a	4.707±0.098^a	4.726±0.076^a	0.461±0.022^a	0.098±0.005^b
SA	1.664±0.029^d	0.473±0.028^d	2.137±0.057^d	3.518±0.066^c	0.242±0.019^d	0.113±0.004^a
MWCNTs+SA	1.929±0.046^c	0.534±0.037^c	2.463±0.064^c	3.612±0.087^b	0.295±0.022^c	0.120±0.003^a

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

3.3. Xanthophyll cycle components (A,V,Z) and ratio

There are three kinds of lutein in the protein complex of leaf thylakoid membrane, one is zeaxanthin (Z, zeaxanthin), the other is epoxy zeaxanthin (A, anthraxanthin), and the third is violaxanthin (V, violaxanthin). These three kinds of lutein Z, A and V can conduct mutual transformation and circulation through epoxidation and de epoxidation reaction. The whole process is also called xanthophyll cycle.^57,58 As shown in Table 3, compared with CK, the content of V, A, Z and VAZ in rice leaves treated with MWCNTs and the xanthophyll cycle capacity increased. Taking 120 h after MWCNTs treatment as an example, the content of V, A, Z and VAZ and the value of (A+Z)/(V+A+Z) in leaves increased by 8.6%, 48.3%, 33.6%, 14% and 21.8% respectively compared with CK. Under saline alkali stress, the content of V and the total content of V+A+Z in the xanthophyll cycle of leaves decreased significantly, while the content of A and Z increased. For example, under saline alkali stress, the V content and the total V+A+Z content of leaves reached 22.23 mmol.mol⁻¹chl and 40.93 mmol.mol⁻¹chl respectively. The content of A and Z showed an increasing trend. The content of A and Z reached 6.42 mmol.mol⁻¹chl and 12.28 mmol.mol⁻¹chl, respectively. The change of above content promoted the enhancement of xanthophyll cycle (A+Z)/(V+A+Z) in rice leaves under saline alkali stress. Compared with SA treatment, the contents of V, A, Z, VAZ and (A+Z)/(V+A+Z) in leaves under MWCNTs+SA treatment increased by 27%, 88.3%, 132.3%, 68.2% and 29.1% respectively, which indicated that MWCNTs could significantly improve the proportion of pigments in lutein cycle and their physiological transformation process under saline alkali stress in rice (Table 3).

Table 3. Effect of MWCNTs on xanthophyll cycle components (A,V,Z) and ratio under saline-alkali stress in rice (120 h).

Treatments	Violaxanthin (V) (mmol.mol^−1chl)	Antheraxanthin (A) (mmol.mol^−1chl)	Zeaxanthin (Z) (mmol.mol^−1chl)	V+A+Z (mmol.mol^−1chl)	(A+Z)/(V+A+Z)
CK	42.51±0.25^b	3.21±0.16^d	6.07±0.15^d	51.79±0.23^c	0.179±0.12^c
MWCNTs	46.15±0.13^a	4.76±0.14^c	8.11±0.19^c	59.02±0.24^b	0.218±0.16^c
SA	22.23±0.21^d	6.42±0.12^b	12.28±0.24^b	40.93±0.18^d	0.457±0.21^b
MWCNTs+SA	28.24±0.27^c	12.09±0.15^a	28.53±0.16^a	68.86±0.15^a	0.590±0.14^a

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

3.4. Gas exchange parameters (P_n, g_s, T_r, WUE, L_s, C_i)

Table 4 shows that t P_n of leaves presents a significant downward trend after 120 h of saline-alkali stress treatment. Compared with SA treatment, MWCNTs+SA treatment significantly improved the photosynthetic capacity of leaves under stress conditions, and P_n increased by 56.47%. Compared with CK, the P_n parameter value of leaves treated with MWCNTs increased by 56.47%. The values of g_s,T_r, L_s and C_i show a similar trend to P_n. Compared with CK, g_s,T_r, L_s and C_i of leaves treated with MWCNTs increased by 3.26%, 2.1%, 9.87% and 8.79%, respectively, of which Ls increased significantly. Compared with SA treatment, the values of g_s,T_r, L_s and C_i of leaves treated with MWCNTs+SA increased by 20.9%, 44.09%, 37.8% and 30.8%, respectively, of which Tr increased significantly. Compared with CK, the WUE values of MWCNTs, SA and MWCNTs+SA treatments increased by 11.9%, 26.1% and 66.7% respectively, which indicates that MWCNTs can help to improve the water use efficiency of leaves under saline-alkali stress, and the improvement of water use efficiency can help to increase the photosynthetic capacity of rice leaves is particularly important, so as to ensure the normal progress of leaf photosynthetic capacity (Table 4).

Table 4. Effect of MWCNTs on gas exchange parameters (P_n,g_s,T_r,WUE,L_s,C_i) under saline-alkali stress in rice (120 h).

Treatments	Pn	gs	Tr	WUE	Ls	Ci
CK	13.26±0.22^a	0.92±0.05^a	5.26±0.25^a	3.45±0.05^c	0.81±0.04^a	225.3±6.25^a
MWCNTs	14.87±0.14^a	0.95±0.06^a	5.37±0.24^a	3.86±0.04^c	0.89±0.05^a	245.1±6.32^a
SA	7.26±0.25^c	0.62±0.04^c	1.86±0.13^c	4.35±0.03^b	0.45±0.06^c	135.6±5.24^c
MWCNTs+SA	11.36±0.16^b	0.75±0.05^b	2.68±0.15^b	5.75±0.04^a	0.62±0.04^b	177.4±5.85^b

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

3.5. Chlorophyll fluorescence parameters (F_m, F_v/F_m, ФPSII, ETR, qP, NPQ)

As shown in Table 5, compared with CK, MWCNTs treatment increased F_v/F_m and Ф PSII, and F_v/F_m and Ф PSII increased by 7.6% and 21.33% respectively, and the difference reached a very significant level, which is extremely beneficial to the comprehensive improvement of leaf photosynthetic capacity. Compared with CK, the F_m, ETR and qP after MWCNTs treatment increased by 4.2%, 1.59% and 1.08% respectively, and the difference was not significant. This indicates that the improvement of photosynthetic capacity of rice leaves under saline-alkali stress is mainly through the increase of F_v/F_m and Ф PSII. Compared with SA treatment, the chlorophyll fluorescence characteristics of rice leaves were improved after MWCNTs+SA treatment, F_m, F_v/F_m, ФPSII, ETR, qP and NPQ increased by 72.3%, 20.9%, 54.8%, 48.7%, 27.1% and 39.7% respectively (Table 5).

Table 5. Effect of MWCNTs on chlorophyll fluorescence parameters (F_m, F_v/F_m, ФPSII, ETR, qP, NPQ) under saline-alkali stress in rice (120 h).

Treatments	Fm	Fv/Fm	ФPSII	ETR	qP	NPQ
CK	1.65±0.02^a	0.92±0.02^b	0.75±0.02^b	98.65±1.25^a	0.92±0.02^a	0.45±0.01^c
MWCNTs	1.72±0.03^a	0.99±0.02^a	0.91±0.01^a	100.21±1.65^a	0.93±0.02^a	0.52±0.02^c
SA	0.65±0.01^c	0.67±0.01^d	0.31±0.02^d	53.68±0.85^c	0.41±0.01^c	0.68±0.01^b
MWCNTs+SA	1.12±0.02^b	0.81±0.02^c	0.48±0.01^c	79.84±1.28^b	0.58±0.02^b	0.95±0.01^a

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

3.6. Activities of RUBPCase and PEPCase in leaves

The enzyme activities of ribulose 1,5-diphosphate carboxylase (RUBPCase) and phosphoenolpyruvate carboxylase (PEPCase) can directly determine the CO₂ fixation capacity of leaves.⁵⁹ Table 6 shows that compared with CK, the activities of RUBPCase and PEPCase in leaves treated with MWCNTs increased. Taking 48 h and 120 h after treatment as an example, the RUBPCase enzyme activity after MWCNTs treatment increased by 5.2% and 9.78% respectively compared with CK, and the PEPCase enzyme activity increased by 4.68% and 12.8% respectively compared with CK. Compared with SA treatment, the photosynthetic enzyme activity of leaves after MWCNTs+SA treatment increased, which contributed to the carbon assimilation process of leaves under saline-alkali stress, and directly promoted the photosynthesis and material accumulation of leaves. At 24 h, 48 h, 72 h, 96 h and 120 h after treatment, the RUBPCase enzyme activity after MWCNTs treatment increased by 2.77%, 1.35%, 4.75%, 9.18% and 11.1% respectively compared with CK, and the PEPCase enzyme activity after MWCNTs treatment increased by 2.48%, 3.87%, 6.21%, 9.52% and 10.42% respectively compared with CK (Table 6).

Table 6. Effect of MWCNTs on activities of RUBPCase and PEPCase under saline-alkali stress in rice.

Treatments	Index	0h	24h	48h	72h	96h	120h
CK	RUBPCase	136.59±2.33^a	145.40±2.32^a	150.77±1.24^b	156.52±1.75^b	154.62±2.33^b	159.89±2.16^b
MWCNTs		136.53±3.12^a	152.43±2.45^a	158.61±1.38^a	159.46±185^a	167.25±1.75^a	175.53±1.75^a
SA		136.43±3.62^a	137.23±3.15^c	141.22±2.31^d	140.37±1.88^d	138.37±2.06^d	129.33±2.34^d
MWCNTs+SA		136.52±2.87^a	141.03±2.04^b	143.12±1.45^c	147.04±1.37^c	151.07±2.16^c	143.68±1.24^c
CK	PEPCase	80.24±1.56^a	84.19±1.24^a	85.73±1.68^b	87.01±0.56^b	87.59±1.24^b	88.05±1.03^b
MWCNTs		80.25±2.04^a	87.92±1.34^a	89.74±1.72^a	92.86±0.78^a	98.59±1.33^a	99.32±0.87^a
SA		80.21±1.24^a	80.64±1.21^c	80.46±1.54^d	79.71±0.49^c	78.68±1.28^d	78.37±0.85^d
MWCNTs+SA		80.23±1.06^a	82.64±1.84^b	83.57±1.64^c	84.66±0.89^b	86.17±1.37^c	86.54±0.75^c

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

3.7. Activities of Sucrose Synthetase (SS) and Sucrose Phosphate Synthase (SPS)

MWCNTs treatment promoted photosynthesis in rice leaves under adverse conditions. Sucrose phosphate synthase (SPS) and sucrose synthase (SS) are involved in sucrose synthesis in the cytoplasm of cells. The decomposition of sucrose into glucose and fructose requires the involvement of invertase, and the co balance of acid invertase (AInv) and alkaline/neutral invertase (A/N-Inv) supports the conversion of sucrose. Table 7 shows that compared to CK, the activities of SS and SPS enzymes in rice leaves after MWCNTs treatment increased, which promoted sucrose synthesis in rice under stress conditions, and promoted intracellular chemical reactions mainly in the direction of sucrose synthesis. For example, compared to CK, the SS enzyme activity of MWCNTs at 24 h, 48 h, 72 h, 96 h, and 120 h after treatment was 20.8%, 25%, 11.7%, 8.5%, and 14.2% higher than that of CK, respectively. Compared to CK, the SPS enzyme activities of MWCNTs treated for 24, 48, 72, 96, and 120 hours were 23.3%, 22.8%, 33.4%, 13.6%, and 32.5% higher than CK, respectively. Under salt and alkali stress, MWCNTs treatment promoted the sucrose synthesis ability of rice leaves. Compared with SA, the SS enzyme activity of MWCNTs+SA treatment at 24 h, 48 h, 72 h, 96 h, and 120 h was 11.2%, 28.6%, 27.3%, 22.9%, and 24% higher than that of SA treatment, respectively. Compared with SA, the SPS enzyme activity of MWCNTs+SA treatment at 24 h, 48 h, 72 h, 96 h, and 120 h was 39.5%, 52.3%, 39.8%, 28.1%, and 36.3% higher than that of SA treatment, respectively. The above shows that MWCNTs promote the transportation and distribution of organic matter produced by photosynthesis in rice leaves by increasing the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in rice leaves under salt and alkali stress, which provides basic material protection for the accumulation of rice material under salt and alkali stress (Table 7).

Table 7. Effect of MWCNTs on activities of sucrose synthetase (SS) and sucrose phosphate synthase (SPS) under saline-alkali stress in rice.

Treatments	Index	0h	24h	48h	72h	96h	120h
CK	SS (mol g^−1 FW h^−1)	23.74±0.45^a	25.98±0.63^b	26.26±0.37^d	28.62±0.51^c	26.72±0.37^d	24.11±0.34^c
MWCNTs		24.66±0.24^a	31.38±0.57^a	32.82±0.44^b	31.98±0.34^b	28.98±0.36^c	27.54±0.31^b
SA		24.15±0.65^a	20.10±0.56^c	27.42±0.21^c	26.22±0.27^d	30.06±0.31^b	22.86±0.24^d
MWCNTs+SA		24.42±0.67^a	24.78±0.43^b	33.66±0.53^a	34.98±0.24^a	34.14±0.28^a	30.30±0.26^a
CK	SPS (mol g^−1 FW h^−1)	28.70±0.34^a	30.13±0.57^b	28.01±0.34^c	27.65±0.26^c	29.22±0.33^c	31.78±0.24^b
MWCNTs		28.99±0.32^a	33.49±0.27^a	36.01±0.41^a	35.21±0.33^b	35.92±0.41^a	39.42±0.28^a
SA		27.70±0.67^a	24.48±0.34^c	21.11±0.43^d	26.04±0.37^d	26.44±0.32^d	22.66±0.33^c
MWCNTs+SA		28.72±0.74^a	34.15±0.24^a	32.16±0.38^b	36.41±0.22^a	33.88±0.31^b	30.88±0.38^b

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

3.8. Activities of invertase enzyme (SAInv) and alkalinity/neutral enzyme (A/N-Inv)

Compared with CK, the activities of SAInv and A/N-Inv enzymes in rice leaves significantly decreased after MWCNTs treatment, indicating that MWCNTs treatment can inhibit the decomposition of sucrose and reduce the activities of SAInv and A/N-Inv enzymes in maize seedling leaves. Compared to CK, the SAInv enzyme activity of MWCNTs at 24 h, 48 h, 72 h, 96 h, and 120 h decreased by 22.8%, 30.1%, 35.7%, 27.5%, and 29.4%, respectively, compared to CK. Compared with CK, the A/N-Inv enzyme activity of MWCNTs treated for 24 h, 48 h, 72 h, 96 h, and 120 h decreased by 27.9%, 31.6%, 16.8%, 13.4%, and 16.6%, respectively, compared to CK. Under salt and alkali stress, MWCNTs treatment promoted the sucrose synthesis ability of rice leaves. Compared with SA, the SAInv enzyme activity of MWCNTs+SA treatment decreased by 17.4%, 19.9%, 43.9%, 26.5%, and 26.3%, respectively, at 24 h, 48 h, 72 h, 96 h, and 120 h compared to SA treatment. Compared with SA, the A/N-Inv enzyme activity of MWCNTs+SA treatment decreased by 36.7%, 28.9%, 35.1%, 18.5%, and 18.4%, respectively, at 24 h, 48 h, 72 h, 96 h, and 120 h compared to SA treatment. Based on the results of SS and SPS studies, we believe that exogenous MWCNTs can significantly enhance the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in leaves under cadmium stress, while reducing the activities of soluble acid invertase (SAIV) and alkaline/neutral invertase (A/N-Inv) in maize leaves. This process is conducive to the accumulation of sucrose in maize leaves, Thereby laying a good physiological foundation for the accumulation and transportation of dry matter from leaves to other organs (Table 8).

Table 8. Effect of MWCNTs on invertase enzyme activities (SAInv) and alkalinity/neutral enzyme activities (A/N-Inv) under saline-alkali stress in rice.

Treatments	Index	0h	24h	48h	72h	96h	120h
CK	SAInv (mol g^−1 FW h^−1)	8.71±0.63^a	12.68±0.34^b	14.86±0.37^a	14.17±0.27^b	14.78±0.28^a	15.85±0.42^a
MWCNTs		8.54±0.64^a	9.78±.027^d	10.38±0.42^c	9.11±0.17^c	10.72±0.19^c	11.19±0.31^c
SA		8.55±0.74^a	13.25±0.27^a	11.53±0.37^b	15.21±0.24^d	13.02±0.11^b	13.49±0.42^b
MWCNTs+SA		8.63±0.55^a	10.94±0.14^c	9.23±0.44^d	8.54± 0.27^d	9.57±0.15^d	9.94±0.28^d
CK	A/N-Inv (mol g^−1 FW h^−1)	12.32±0.24^a	19.99±0.32^a	21.48±0.38^b	20.61±0.28^b	17.51±0.12^c	15.98±0.32^b
MWCNTs		12.10±0.33^a	14.41±0.21^b	14.70±0.29^d	17.14±0.41^c	15.17±0.21^d	13.32±0.26^c
SA		12.08±0.41^a	13.17±0.24^c	22.35±0.33^a	22.60±0.48^a	22.85±0.23^a	19.57±0.27^a
MWCNTs+SA		12.53±0.54^a	8.34±0.35^d	15.90±0.37^c	14.66±0.29^d	18.63±0.24^b	15.96±0.36^b

*Means within the same row followed by the different small letters show that there are significant differences at p<.05.

4. Discussion

The regulation of plant growth and development by carbon nanomaterials is a newly emerging research hotspot. The rapid development of nanotechnology has been promoting the transformation of traditional agriculture. The green synthesis technology of nanoparticles has avoided the use of a large number of chemicals, while also reducing the environmental pollution caused by toxic substances generated during the preparation process, causing widespread concern. Previous research on carbon nanomaterials has mainly focused on the possible risks to the environment and human body,⁶⁰ with a focus on their impact on animals or human cells.^61,62 Studies have found that low concentration carbon nanomaterials play a positive role in plant growth and development, mainly in stimulating crop seed germination, promoting plant root elongation, improving the growth rate of callus, and accumulating plant biomass. Other studies have shown that under certain concentrations and exposure conditions, carbon nanomaterials have negative effects on plant growth and development, manifested as tissue damage, growth inhibition, and an increase in ROS (reactive oxygen species), and the negative effects depend on plant species and growth conditions.^63,64 MWCNTs can significantly improve the elongation of wheat root cells, enhance dehydrogenase activity, and ultimately promote rapid root growth and yield improvement.⁶⁵ During the field growth stage, MWCNTs can also be used as plant growth regulators to improve the flowering and seed setting rates of tomatoes and ensure stable crop yields.⁶⁶ Carbon nanotubes can promote rice seed germination, improve root growth and root activity. There are also some controversial conclusions in the experiment of carbon nanomaterials regulating plant growth. For example, some studies suggest that MWCNTs increase the fresh weight and length of wheat roots, but have no significant impact on wheat seed germination rate.^65,67 The germination rate of corn and ryegrass was inhibited at the concentration of 2000 mg/LMWCNTs, but the root length increased.⁶⁸ In summary, previous studies have shown that carbon nanomaterials have a certain dose effect on the regulation of plants. Studies have shown that carbon nanomaterials can regulate plant growth and development by influencing gene expression in plants, mainly by upregulating the expression of some aquaporin genes, promoting the growth of tobacco callus with MWCNTs, and upregulating the expression of aquaporin gene NtPIP1 and CycB related to cytokinesis.⁶⁹ Based on previous studies, it has been speculated that there are three main modes of action of carbon nanomaterials on plants. One is that carbon nanomaterials adsorb on the surface of plant roots through their huge adsorption properties, thereby promoting root elongation of onions and cucumbers;⁷⁰ The second is that carbon nanomaterials enter the plant body to adsorb on the cell wall or enter the cell interior, resulting in a 1.4-fold increase in the cell length of the root zone of the seedlings, and a significant increase in dehydrogenase activity;⁶⁸ Third, carbon nanomaterials act on the rhizosphere environment. Some studies have shown that MWCNTs affect the composition of soil microbial communities.^26,71

100 mg/L cerium oxide nanoparticles can promote soybean growth and increase photosynthetic rate by 54%; However, at a concentration of 500 mg/L, the photosynthetic rate of soybean decreased by about 36%.⁷² Previous studies have shown that the content of chlorophyll a, chlorophyll b, and carotenoids in cotton treated with zinc oxide nanoparticles significantly increased.⁷³ twenty μ After using g/mL silica nanoparticles in Arabidopsis thaliana, the carotenoid content in its leaves increased by 61.7%, and the photosynthetic capacity of chloroplasts increased by 34.3%.⁷⁴ Compared with NaCl treatment, the total chlorophyll content in pumpkin leaves increased by 345%, the chlorophyll degradation rate decreased by 45.13%, and the carbonic anhydrase activity increased by 26.62%.⁷⁵ Copper nanoparticles and silver nanoparticles can enhance somatic embryogenesis and regeneration of wheat mature embryo explants.⁷⁶ In the concentration range of 100 to 1000 mg/L, with the increase in the concentration of copper oxide nanoparticles, the photosynthetic rate, transpiration rate, stomatal conductance, and photosynthetic pigment content of rice gradually decrease, ultimately leading to its inability to perform photosynthesis.⁷⁷ Applying metal nanoparticles can effectively increase the content of corresponding elements in plants, thereby promoting plant growth.⁷⁸ For example, titanium oxide and iron based nanoparticles can delay aging and accelerate cell division by changing plant hormone levels, thereby promoting plant growth. This study shows that MWCNTs treatment can significantly enhance the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) under salt and alkali stress, and reduce the activities of soluble acid invertase (SAInv) and alkaline/neutral invertase (A/N-Inv). It indicates that MWCNTs promote sucrose synthesis while inhibiting sucrose decomposition, thereby promoting the accumulation of sucrose in rice leaves. The modes of long-distance transportation of nanomaterials in crops mainly include xylem and phloem transportation. After being absorbed by plant roots, nanomaterials enter xylem vessels, follow the path of transpiration water, and are transported to the phloem, where they are then transported to the ground through xylem and phloem transportation. Some studies have shown that after being absorbed by plants, some silver nanoparticles accumulate in the root, while others mainly migrate to the xylem in the form of nanoparticles, and then to other plant organs. This movement may occur through the vascular system.^79,80 Treating tobacco cells with different concentrations of MWCNTs can significantly promote tobacco cell growth and induce cell division, cell wall formation, and expression of related genes such as aquaporins.⁶⁹ However, some studies have shown that the accumulation of high concentration nano materials in plants can inhibit plant growth, reduce crop quality, seed germination rate, reduce fresh and dry weight, and root and bud length.⁷⁷ In summary, nanomaterials have advantages and disadvantages for crop growth and development, and attention should be paid to comprehensive evaluation of their safety and risk when applying them. Using scanning electron and fluorescence microscopy, it was confirmed that water-soluble CNTs exist in wheat plants, and CNTs can induce rhizome growth under both light and dark conditions.⁸¹ CNTs induce the absorption efficiency of water and essential Ca and Fe nutrients, thereby promoting plant growth and development.^82,83 The addition of nano carbon has increased the content of available nitrogen, phosphorus, and potassium nutrients in the soil, and promoted the accumulation of nitrogen, phosphorus, potassium, and other nutrients in maize.⁸⁴

A large number of experiments and studies have shown that most nanomaterials have positive and negative effects on rice, while some nanomaterials have no effect on rice. The positive effects on rice include promoting seed germination, growth and development, increasing yield, and upregulating the expression of favorable genes. For example, when CDs-1 was introduced into rice, 500 up-regulated genes and 87 down-regulated genes were found through RNA sequencing.⁸⁵ Most of the negative effects are due to the toxic effects of metal nanomaterials on rice, which have adverse effects on its growth and development, including seed germination. Some materials can inhibit rice growth. For example, a high concentration of NCuO (<50 nm) solution has an inhibitory effect on the germination rate, root length, bud length, and biomass of rice.⁸⁶ There are also some materials that have no effect on rice growth. When rice is simultaneously applied with different concentrations of nano silicon oxide and ordinary silicon oxide materials, it is found that there is no significant effect on rice growth.⁸⁷ In summary, carbon nanotubes can affect the absorption of water by plant seeds and roots, thereby affecting the absorption of nutrients by plants, thereby promoting plant growth and development.⁸⁸ Our results show that MWCNTs can improve the water use ability of roots and leaves, especially the water absorption ability of roots, which provides a guarantee for the improvement of rice biomass and the enhancement of leaf photosynthetic capacity under adverse conditions, which is basically consistent with previous research results. In addition to the positive effects of carbon nanotubes on plant growth and development, previous studies have also found some side effects of carbon nanotubes. In the study of rice, it was found that multi walled carbon nanotubes have a toxic effect on suspension cultured rice cells and cause some cells to agglomerate.^82,89,90 At the same time, it was also found that multi walled carbon nanotubes can lead to delayed flowering and decreased seed setting rate in rice, resulting in reduced yield.⁹¹ Therefore, the impact of carbon nanotubes on plant growth and development is complex and variable, which mainly depends on the type, concentration, particle size, and plant genotype of carbon nanotubes.^23,92–94 Carbon nanotubes can affect the growth and development of plants, which are regulated to some extent by gene expression. Therefore, carbon nanotubes may affect the expression of related genes, thereby affecting the growth and development of plants. Studies have shown that multi-walled carbon nanotubes can induce the expression of an unknown gene in tomato leaves and roots, causing the transcription level of stress-related genes, including the aquaporin gene LeAqp2, to be upregulated, as well as the expression level of genes related to cell division and differentiation.⁹⁵ Low concentrations of multi walled carbon nanotubes can also increase the transcriptional water content of the tobacco aquaporin gene NtPIP1 and the NtLRX1 gene related to cell differentiation.⁶⁹ Carbon nanotubes can inhibit the expression of genes related to root hair development in Arabidopsis, upregulate the transcription levels of some genes related to stress, the key gene for ABA synthesis, NCED, and auxin response factors in plants, and activate the expression of hormone transport related genes.⁹⁶ To sum up, carbon nanotubes can affect the expression of a series of genes and activate some stress tolerance related genes, but further research in this area remains to be carried out.

Nanomaterials can significantly promote photosynthesis and greatly improve spinach growth, and it is speculated that the promotion effect of nanomaterials treatment on spinach growth may be closely related to changes in nitrogen metabolism. It is also possible to observe an instantaneous increase in net photosynthetic rate and stomatal conductance, and a decrease in non photochemical quenching.^97–100 Based on the analysis of the impact of MWCNTs on plant photosynthesis, MWCNTs can increase net photosynthetic rate and reduce heat dissipation, which has guiding significance for plant growth under salt and alkali stress conditions and for improving the yield and quality of crops. Our research results show that the photosynthetic rate (Pn), stomatal conductance (g_s), and transpiration rate (T_r) of leaves treated with MWCNTs increased significantly, and the photochemical quenching value (qP), photochemical quantum efficiency value (F_v/Fm), and electron transfer rate value (ETR) of chlorophyll fluorescence parameters increased significantly, which is beneficial to the improvement of the PSII photosynthetic system. MWCNTs treatment promoted the increase of photosynthetic pigment content in leaves under salt and alkali stress, improved the ratio of Chla and Chlb parameters, increased the activities of key photosynthetic enzymes (RUBPCase and PEPCase) in leaves, increased the value of total lutein cycle pool (VAZ), and significantly enhanced the deepoxidation effect of lutein cycle (DEPS), which can effectively alleviate the stomatal and non stomatal constraints on leaf photosynthesis caused by salt and alkali stress. Our results suggest that MWCNTs can promote rice growth under salt and alkali stress by simultaneously upregulating energy storage in photosynthesis and downregulating energy consumption in metabolism. The mixed application of carbon nanomaterials and fertilizers can promote the growth and development of various crops, but it may also have a inhibitory effect under certain conditions, which is related to the type and concentration of materials, as well as plant varieties and growth conditions.¹⁰¹ At present, there is still a lack of effective research on the interaction laws and regulatory mechanisms between carbon nanomaterials and crops. Therefore, the ways in which carbon nanomaterials affect crops, their entry pathways, and their effects on gene expression and signal transduction in crops will become the focus of future research. Although carbon nanomaterials have shown great potential in regulating plant growth and development, their mechanism of action is unclear. The interaction between carbon nanomaterials and plants lacks relevant reports on more plant species and materials. There is a lack of effective research on the pathways and regulatory mechanisms of carbon nanomaterials in promoting plant growth. Therefore, the way in which carbon nanomaterials act on plants, their entry pathways, and their effects on plant gene expression and signal transduction will be the focus of future mechanism research. The above research will help to better understand the response mechanism of nanomaterials in plant growth and development, and better apply them to agricultural production.

5. Conclusions

This study indicates that MWCNTs have a positive effect on promoting rice growth under salt and alkali stress. MWCNTs can improve the water use ability of roots and leaves, especially the water absorption ability of roots, which provides a guarantee for the improvement of rice biomass and the enhancement of leaf photosynthetic capacity under adverse conditions. MWCNTs treatment promoted the increase of photosynthetic capacity of leaves under salt and alkali stress, with significant increases in Pn, gs, and Tr values, and significant increases in chlorophyll fluorescence parameters such as qP, F_v/F_m and ETR, which is beneficial to the improvement of PSII photosynthetic system. MWCNTs treatment promoted the increase of photosynthetic pigment content in leaves under salt and alkali stress, improved the ratio of Chla and Chlb parameters, increased the activities of key photosynthetic enzymes (RUBPCase and PEPCase) in leaves, increased the value of total lutein cycle pool (V+A+Z), and significantly enhanced the de epoxidation effect of lutein cycle, which can effectively alleviate the stomatal and non stomatal constraints on photosynthesis in rice leaves under salt and alkali stress

Author contributions

Conceptualization, P.Y.; software, Z.X.; validation, H.L.; formal analysis, Z.X.; investigation, Y.Y.; data curation, S.Z. and D.G.; writing original draft preparation, Z.X.; writing – review and editing, Z.X. and P.Y.; project administration, P.Y; funding acquisition, P.Y. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was funded by the Scientific research funds of Heilongjiang provincial research institutes (CZKYF2021-2-A003）and Scientific research funds of Heilongjiang provincial research institutes (CZKYF2022-1-C042); Science and technology Project of Heilongjiang Academy of Agricultural Sciences (CX23YQ15 and 2021YYYF053); China Agriculture Research System “Wuchang Comprehensive Experimental Station”(CARS-01-59).