Method for evaluating drought resistance of tomatoes under drought stress based on root pressure research

Abstract

The invention discloses a method for evaluating drought resistance of tomatoes under drought stress based on root pressure research, and relates to the technical field of agriculture. The method comprises the following steps: (1) carrying out morphological dissection on root systems of large-fruit tomatoes and cherry tomatoes; (2) drought stress treatment and rehydration; (3) exogenous ABA treatment: removing overground parts to eliminate overground transpiration, exposing roots in a 50M ABA solution for 1 hour, and determining root pressure and ROS content of root tips; and (4) comprehensively analyzing the response of the root system of the tomato under the drought stress according to the form of the tomato leaf, the root pressure and the substances in the signal channel, and judging the drought resistance of the root system under the drought stress according to the form of the partial leaf on the ground. According to the technical scheme, the drought resistance of the two tomatoes is evaluated through various indexes under drought stress, and a theoretical basis is provided for subsequent drought-resistant breeding and water and fertilizer management.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, and in particular to a method for evaluating the drought resistance of tomatoes under drought stress based on root pressure studies. Background Technology

[0002] Drought is a global climate problem that severely restricts agricultural development (Li et al., 2021). The root system's ability to absorb water directly determines the water status within plant tissues, and the root system's water absorption capacity under drought stress is crucial for plant drought resistance (Zhu JK, 2016; Cai et al., 2022). Water absorption mainly occurs in the root hair zone and enters the xylem through three pathways. First, the apoplast pathway involves rapid transport of water through the cell walls and intercellular spaces of the epidermis, exodermis, and cortex. Second, the symplast pathway refers to water entering the cell and being absorbed through adjacent plasmodesmata without transmembrane transport. Finally, the transcellular pathway requires water to cross the cytoplasmic membrane; each cell requires crossing two membranes, and sometimes even the vacuolar membrane. The permeability of the membranes determines the rate of water uptake. Water reaches the endodermis, but due to the thickened cell walls both horizontally and vertically, water cannot cross the cell walls and intercellular spaces. Therefore, water transport through the endodermis occurs via only two pathways: water from the symplast enters the endodermal cells through plasmodesmata, and water from the apoplast must pass through the plasma membrane of the endodermis to enter. Water passing through the endodermis reaches the parenchyma cells of the xylem and enters the vessels or tracheids. Finally, it is transported upwards along the osmotic potential difference to the next higher root level and eventually reaches the stem and leaves of the above-ground parts (Tardieu et al., 2017).

[0003] Plants absorb soil moisture through their roots and transport it vertically upwards over distances of several meters or even hundreds of meters, primarily driven by transpiration pull (Cao et al., 2012). Root pressure also propels water upwards through xylem or tracheids (Zachary, 2009). Due to root physiological activities, ions and soluble organic matter in the cortex continuously pass through the endodermis into the stele, creating an osmotic gradient. Driven by this osmotic gradient, water flows from the outside into the xylem or tracheids within the stele, accumulating and generating positive hydrostatic pressure (Yang et al., 2024). Sap flow and gavage are the best evidence that root pressure is a lifting force that causes sap flow upwards (Taiz et al., 2015). Root pressure has significant biological importance in multiple plant science research fields, including plant ecology, plant physiology, agronomy, and horticulture (Yang et al., 2012; Zhao et al., 2017; Nardini et al., 2018). Studies have reported that roots can rapidly synthesize abscisic acid (ABA) under drought stress, which participates in root water absorption. Resinogen (ROS) is also believed to be involved in ABA-mediated root water uptake (Guzel et al., 2016). Research shows that drought-resistant varieties have higher root pressure values ​​under drought stress, and the root pressure recovery rate of drought-resistant varieties is 30%–50% faster than that of sensitive varieties after the drought stress is relieved.

[0004] Because roots grow underground, sampling and research techniques present certain difficulties, so most studies focus on the aboveground parts of plants. However, root pressure measurement techniques are relatively mature and can be categorized into direct and indirect methods. Direct measurement includes both damaging and non-damaging techniques. Damaging techniques include manometer methods, root pressure probe techniques, and root pressure chamber techniques, while non-damaging techniques include isobaric methods, xylem pressure probe techniques, and cell pressure probe techniques. Indirect measurement involves using non-damaging techniques to measure minute changes in leaf thickness or stem diameter at night, then comparing the actual measurements with predicted values ​​using relevant models to calculate the root pressure (Zhang et al., 2020).

[0005] Tomato (Solanum lycopersicum L.) is an annual herbaceous plant belonging to the genus Solanum in the family Solanaceae. Due to its excellent taste, attractive color and shape, and rich content of carotene, vitamin C, B vitamins, and antioxidants, it is widely cultivated globally as both a vegetable and a fruit, possessing health and horticultural value (Vats et al., 2022). Furthermore, as the model plant of the Solanaceae family, the tomato holds a similar position in plant research as Arabidopsis thaliana and rice. Tomatoes are generally divided into large-fruited tomatoes and small-fruited tomatoes. Large-fruited tomatoes are what we commonly call tomatoes, while small tomatoes, scientifically known as cherry tomatoes, are also called "sacred tomatoes" or "mini persimmons." Although they belong to the same species and share the same Latin name, their appearance and nutritional composition differ (Yu Dinglang & Tan Shuming, 2018).

[0006] Tomato production is severely affected by abiotic stresses (drought, low light, high temperature, and salinity), resulting in yield losses of approximately 70% depending on the severity and duration of the stress. Drought is the most destructive abiotic stress, and tomatoes are highly sensitive to it (Krishna et al., 2022). Gansu Province, located in Northwest China, is an inland non-monsoon region with annual precipitation below 400 mm and dry air. Rainfall becomes a limiting factor for crops. To cultivate more drought-resistant tomatoes, it is essential to determine suitable water conditions for growth and to breed tomato germplasm adapted to the region's climate. Current research on tomatoes mainly focuses on cultivation and component analysis, while drought resistance research primarily investigates signaling pathways (Liu et al., 2021; Lin et al., 2022). Traditional drought resistance research mainly focuses on the relationship between soil moisture content and above-ground morphology. However, soil moisture content and plant growth status are not positively correlated. For example, when ferns grown in southern regions are transplanted to Qingyang, even with high soil moisture content, their leaves will gradually wither and the plants will eventually die. This is because the root system's water absorption capacity is less than the leaf transpiration rate, leading to water shortage. Currently, there is limited research on tomato drought resistance from the perspective of root water absorption.

[0007] There is an urgent need in the existing technology for a method to evaluate the drought resistance of tomatoes under drought stress based on root pressure research. Summary of the Invention

[0008] In view of this, this invention provides a method for evaluating the drought resistance of tomatoes under drought stress based on root pressure research. It utilizes plant physiology techniques to study the root morphology and root pressure of large-fruited tomatoes and cherry tomatoes: 1. To explore the relationship between soil moisture content, root pressure, and tomato growth status. 2. By measuring root pressure and ROS content in root tip cells of both tomato varieties after drought treatment and exogenous ABA treatment, the relationship between root pressure and ROS in tomatoes under drought stress is revealed. 3. The drought resistance of the two tomato varieties is assessed using various indicators under drought stress, providing a theoretical basis for subsequent drought-resistant breeding and water and fertilizer management.

[0009] To achieve the above objectives, the present invention mainly provides the following technical solutions:

[0010] On one hand, embodiments of the present invention provide a method for evaluating the drought resistance of tomatoes under drought stress based on root pressure studies, the method comprising the following steps:

[0011] (1) Planting

[0012] Two types of tomato seedlings were selected for the experiment. The study showed that root pressure during the seedling stage became the main driving force for water absorption. The soil moisture content of the control group was controlled at 65%-70%. Ten healthy seedlings of the same size for each tomato were selected for pot planting.

[0013] (2) Drought stress treatment

[0014] Four water treatments were set up: normal watering (control), 75% of normal watering (mild stress), 50% of normal watering (moderate stress), and 25% of normal watering (severe stress). Soil moisture content, root characteristic parameters, root tip cell ROS content, root pressure and other indicators were measured.

[0015] Post-drought stress rehydration treatment

[0016] When the drought stress reaches a severe level (5 days of drought treatment), rehydration begins, root pressure is measured immediately, and root characteristic indicators are measured one week later.

[0017] (3) Exogenous ABA treatment

[0018] The aboveground parts were removed to eliminate aboveground transpiration, and the roots were exposed to 50 µM ABA solution for 1 h to measure root pressure and root tip ROS content.

[0019] (4) Based on the comprehensive analysis of the substances in the two tomato root pressure and signaling pathways, the root response of tomatoes under drought stress was analyzed. At the same time, the ability of the root system to absorb water under drought stress, i.e. drought resistance, was judged based on root pressure, which provides technical support for subsequent water management and drought-resistant breeding of tomatoes.

[0020] Furthermore, the tomatoes mentioned are large-fruited tomatoes and cherry tomatoes.

[0021] Furthermore, the various indicators of the root mentioned in step (1) include the root type, root length, and root density.

[0022] The root system Epson V800 was used to analyze the root type, root length, and root density.

[0023] Further, in step (3), the ABA content determination is specifically as follows: The ABA determination is based on the method of Verslues (2017), with slight modifications. The mature part of the tomato root collected after treatment is rapidly ground in liquid nitrogen, extracted with 8 ml of mixed extraction solution (80% methanol with added BHT), and incubated overnight at -20℃. Then, it is centrifuged at 10000×g for 10 minutes at 4℃, and the supernatant is collected. Extract the residue again with 4 ml of extractant, combine the two supernatants, and evaporate with nitrogen to 1 / 3 of the original volume using a nitrogen evaporator (HP5106GD, Shanghai Oriental Analytical Instruments Co., Ltd., Shanghai, China). Adjust the pH of the solution to 8.0 with 1 mol / L sodium dihydrogen phosphate, add an equal volume of petroleum ether, and extract three times. Discard the ester phase, add polyvinylpyrrolidone (PVPP, 0.1 g) (for adsorbing phenolic substances and pigments), and place the sample on a temperature-controlled shaker at 130 r / min for 30 minutes. Filter the PVPP, adjust the pH of the extract to 3.0, and extract three times with an equal volume of ethyl acetate. Combine the extracts, approximately 9 ml. Evaporate the extract dry with nitrogen, then dissolve the residue in 4 ml of phosphate buffer solution (pH=3), and pass it through a reverse-phase C18 solid-phase extraction column (Simon Aldrich, Germany; C18 / 200 mg / 3 ml). Elute the column eluent dry with nitrogen and finally dissolve it in 1 ml of methanol. The dissolved sample was filtered through a 0.22 µm microporous membrane, and the ABA content was determined using high-performance liquid chromatography (HPLC) (OrbiTrap Fusion LUMOS, ThermoFisher, San Jose, California, USA). ABA content (ng / g) -1 (dry weight) represents the ABA content per unit dry weight.

[0024] Further, in step (3), the root pressure measurement method is as follows: The method is slightly modified from that of Yang Shijian et al. (2012). First, the above-ground parts of the tomato are cut off to avoid the influence of transpiration on root pressure. Then, the stem is wrapped with sealing film, and a rubber hose (3 mm inner diameter) filled with deaeration water is used to connect the tomato stem to the pressure sensor (PX26-30GV, OMEGA, Stamford, USA). The signal output of the pressure sensor is connected to a data acquisition unit (CR1000x, Campbell Scientific, Logan, USA). Data is collected every 10 seconds, and the average value is recorded every 1 minute. The data acquisition unit can simultaneously measure battery voltage and room temperature. Before testing, the instrument needs to be calibrated. The voltage value is recorded by water pressure at five different heights. The pressure sensor output is in mV, which needs to be converted to kPa.

[0025] Further, in step (3), the ROS content determination method is basically based on the description of Arnaud et al. (2017), using the fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma, USA) to determine the production of ROS in tomato root cells. After the roots were exposed to 50 µM ABA solution for 1 h, the mature root tip was prepared and immediately examined using a laser scanning confocal microscopy (LSCM). The excitation wavelength was 488 nm, and the emission wavelength was 505-530 nm. The fluorescence intensity of the acquired images was analyzed using ImageJ software (https: / / imagej.nih.gov / ij / ) (V1.8.0.112, USA). The sites of fluorescence production were analyzed, and the magnitude of the increase in ROS fluorescence intensity was calculated. This technique can monitor which tissues and cells are involved in ROS synthesis and can determine the degree of ROS increase.

[0026] This experiment detected ROS fluorescence signals in the endodermis of the root hair zone, and the parenchyma cells of the cortex showed obvious ROS fluorescence signals.

[0027] When subjected to severe stress (soil moisture content of 25% as control), most plants die, with only a small number surviving and exhibiting root pressure, which provides technical support for breeding highly drought-resistant tomato plants.

[0028] Preferably, when the soil moisture content is 25% of the control, the root pressure of the large-fruited tomato is 5.2 ± 1.3 kPa.

[0029] Preferably, when the soil moisture content is 25% of the control, the root pressure of the cherry tomato is 3.1 ± 0.9 kPa.

[0030] On the other hand, embodiments of the present invention provide the application of the method in the tomato cultivation process.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] Root pressure is the result of a series of metabolic processes in the root system and is the main driving force for plants to actively absorb water, especially during droughts and at night. Plants rely on root pressure to absorb water to replenish the water in their tissue cells. Traditional drought resistance experiments mainly rely on visual observation of plant morphology to determine drought resistance, and research on analyzing plant drought resistance from the perspective of root pressure is almost non-existent. This study uses root pressure measurements under different water stresses to determine the drought resistance of plants, which is of great significance for water management in tomato cultivation and the breeding of drought-resistant varieties. Attached Figure Description

[0033] Figure 1 This is a technical roadmap of the technical solution of this invention;

[0034] Figure 2 The effect of ABA treatment on root pressure in two types of tomatoes;

[0035] Figure 3 The effect of ABA treatment on ROS content in the root hair zone of two tomato varieties;

[0036] Figure 4 The effect of moderate stress on the ABA content in the roots of two tomato species. Detailed Implementation

[0037] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following preferred embodiments are provided to describe in detail the specific implementation methods, technical solutions, features, and effects according to the present invention. Specific features, structures, or characteristics in the various embodiments described below can be combined in any suitable form.

[0038] This invention uses large-fruited tomatoes and cherry tomatoes as research subjects. It analyzes the root structure characteristics of these two tomato varieties, the relationship between soil moisture and root pressure, and measures ABA and ROS content and root pressure under drought stress. The response levels within root cells are used to reflect the drought resistance of the two tomato varieties. Ultimately, this research aims to improve water management and screen for drought-resistant tomato plants. Figure 1 As shown, the specific research plan is as follows:

[0039] (1) Morphological anatomy of the roots of large-fruited tomatoes and cherry tomatoes

[0040] The root system analysis system is used to statistically analyze indicators such as root type, root length, and root density.

[0041] (2) Drought stress treatment

[0042] Watering was stopped for 1, 2, 3, and 5 days, and soil moisture content, tomato leaf water potential, root ABA content, root tip cell ROS content, root pressure, and other indicators were measured.

[0043] ABA content determination: The ABA determination method was based on Verslues (2017) with slight modifications. The mature root portion of the treated tomato was rapidly ground in liquid nitrogen and extracted with 8 ml of a mixed extract (80% methanol with added BHT) at -20°C overnight. Then, it was centrifuged at 10000×g for 10 minutes at 4°C, and the supernatant was collected. Extract the residue again with 4 ml of extractant, combine the two supernatants, and evaporate with nitrogen to 1 / 3 of the original volume using a nitrogen evaporator (HP5106GD, Shanghai Oriental Analytical Instruments Co., Ltd., Shanghai, China). Adjust the pH of the solution to 8.0 with 1 mol / L sodium dihydrogen phosphate, add an equal volume of petroleum ether, and extract three times. Discard the ester phase, add polyvinylpyrrolidone (PVPP, 0.1 g) (for adsorbing phenolic substances and pigments), and place the sample on a temperature-controlled shaker at 130 r / min for 30 minutes. Filter the PVPP, adjust the pH of the extract to 3.0, and extract three times with an equal volume of ethyl acetate. Combine the extracts, approximately 9 ml. Evaporate the extract dry with nitrogen, then dissolve the residue in 4 ml of phosphate buffer solution (pH=3) and pass it through a reverse-phase C18 solid-phase extraction column (Simon Aldrich, Germany; C18 / 200 mg / 3 ml). Elute the column eluent dry with nitrogen and finally dissolve it in 1 ml of methanol. The dissolved sample was filtered through a 0.22 µm microporous membrane, and the ABA content was determined using high-performance liquid chromatography (HPLC) (OrbiTrap Fusion LUMOS, Thermo Fisher, San Jose, California, USA). ABA content (ng / g) -1 (dry weight) represents the ABA content per unit dry weight.

[0044] (3) Exogenous ABA treatment

[0045] The aboveground parts were removed to eliminate aboveground transpiration, and the roots were exposed to 50 µM ABA solution for 1 h to measure root pressure and root tip ROS content.

[0046] Root pressure measurement method: The method was slightly modified from that of Yang Shijian et al. (2012). A rubber hose (3 mm inner diameter) filled with deaerated water was used to connect the stem to the pressure sensor (PX26-30GV, OMEGA, Stamford, USA). The signal output of the pressure sensor was connected to a data logger (CR1000x, Campbell Scientific, Logan, USA). Data was collected every 10 seconds, and the average value was recorded every 1 minute. The data logger could simultaneously measure battery voltage and room temperature. Before testing, the instrument needed to be calibrated. Voltage values ​​were recorded by measuring water pressure at five different heights. The pressure sensor output was in mV, which needed to be converted to kPa.

[0047] ROS content determination: The method was basically based on the description of Arnaud et al. (2017), using the fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma, USA) to determine ROS production in tomato root cells. Root tips were prepared after root exposure to 50 µM ABA solution for 1 h and immediately examined using laser scanning confocal microscopy (LSCM). The excitation wavelength was 488 nm, and the emission wavelength was 505-530 nm. The fluorescence intensity of the acquired images was analyzed using ImageJ software (https: / / imagej.nih.gov / ij / ) (V1.8.0.112, USA).

[0048] (4) Based on the morphology, root pressure and substances in the signaling pathway of tomato roots, the root response of two types of tomatoes under drought stress was comprehensively analyzed. At the same time, the ability of the roots to absorb water under drought stress, i.e. drought resistance, was judged based on root pressure, so as to provide technical support for the subsequent water management and drought-resistant breeding of tomatoes.

[0049] Example

[0050] Plant material cultivation and selection: For each treatment experiment, 30 healthy large tomato seedlings and 30 small tomato seedlings of uniform size were selected, with 10 seedlings per replicate as the sample size, and the experiment was repeated 3 times (n=3). The experimental stage was from the seedling stage to the initial flowering stage.

[0051] Drought stress treatment: A pot experiment was conducted with four water treatments: normal watering (control: soil moisture content 65%-70%), 75% of normal watering (mild stress), 50% of normal watering (moderate stress), and 25% of normal watering (severe stress).

[0052] Effects of drought stress on root growth: By measuring root length and root volume after different degrees of drought stress, the study found that mild stress promoted the growth of root systems in both tomato species, with a certain increase in taproot length and total root volume, while moderate and severe stress inhibited root growth (Tables 3 and 4).

[0053]

[0054]

[0055] Effects of drought stress on plant morphology: By observing the plant morphology after different degrees of drought stress, it was found that mild stress had little effect on the morphology of the two tomato plants, and most of them grew normally. Under moderate stress, most plants survived but showed mild stress symptoms. Under severe stress, a very small number of plants survived but showed severe stress symptoms, such as leaf curling and growth cessation. Moreover, they could recover their normal morphology after rehydration (Table 1, Table 2).

[0056] The effect of drought stress on root pressure: Root pressure was measured after different degrees of drought stress. It was found that root pressure increased in both types of tomato plants under mild stress, decreased significantly under moderate stress, and was absent in most plants under severe stress. A small number of surviving plants had low root pressure, but the surviving plants recovered to normal root pressure after rehydration (Tables 1 and 2). This indicates that this method can screen for drought-resistant individuals, while the traditional method of judging plant drought resistance by visual observation of plant morphology has certain drawbacks.

[0057] ABA content determination: Under moderate stress, surviving tomato seedlings were selected, and the ABA content of the root mature zone was determined. The specific method is described in the experimental methods section above. Results showed that the ABA content in the roots of both tomato species significantly increased after moderate stress. Figure 4 ).

[0058] ABA solution treatment: After treatment with 50 µM exogenous ABA, the root pressure values ​​of the two tomato varieties increased significantly compared to the control varieties. Figure 2 Furthermore, ROS fluorescence intensity was significantly increased in the endodermis of the root hair zone after ABA treatment. Figure 3 ).

[0059] Experiments using ABA content measurement and exogenous ABA treatment demonstrated that ABA participates in root pressure generation under drought stress. This experimental scheme is a method for evaluating the drought resistance of two tomato varieties under drought stress based on root pressure research. This innovative method utilizes root pressure in conjunction with other indicators to comprehensively analyze drought resistance, providing technical support for the breeding of two drought-resistant tomato varieties.

[0060] Any aspects not covered in the embodiments of this invention can be selected from the prior art by those skilled in the art.

[0061] The above descriptions are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the above claims.

Claims

1. A method for evaluating drought resistance of tomato under drought stress based on root pressure, characterized in that, The method comprises the following steps: (1) planting Two kinds of tomato seedlings are selected for the experiment. The results show that the root pressure becomes the main water absorption power in the seedling stage. The soil water content of the control group is controlled at 65%-70%. Ten healthy seedlings with consistent size are selected for each treatment; (2) drought stress treatment Four water treatments are set, namely normal watering, 75% of normal watering amount, 50% of normal watering amount, and 25% of normal watering amount. The soil water content, tomato leaf water potential, root ABA content, root tip cell ROS content, and root pressure are measured; (3) rehydration treatment after drought stress When the drought stress reaches 50% of the normal watering amount, rehydration is started. The root pressure is measured; (3) exogenous ABA treatment The aboveground part is removed to eliminate the aboveground transpiration. The root pressure and root tip ROS content are measured after exposing the root to 50 µM ABA solution for 1 h; (4) The response of the tomato root system under drought stress is comprehensively analyzed according to the morphology of the tomato leaf, the root pressure, and the substances in the signal pathway. The root water absorption capacity under drought stress is determined according to the root pressure.

2. The method for evaluating drought resistance of tomato under drought stress based on root pressure research according to claim 1, characterized in that, The tomato is large fruit tomato and cherry tomato.

3. The method for evaluating drought resistance of tomato under drought stress based on root pressure research according to claim 2, characterized in that, In step (3), the ABA content is determined by taking samples from the phloem of the mature zone of the tomato root.

4. The method for evaluating drought resistance of tomato under drought stress based on root pressure research according to claim 3, characterized in that, When the soil water content is 50% of the control, the root pressure of the large fruit tomato is 16.6±2.5 kpa.

5. The method for evaluating drought resistance of tomato under drought stress based on root pressure research according to claim 3, characterized in that, When the soil water content is 50% of the control, the root pressure of the cherry tomato is 12.5±2.0 kpa.

6. The method according to any one of claims 1-5 is applied in the process of tomato planting.

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