Use of mannose in enhancing plant root development in petroleum-contaminated soils

Abstract

The application discloses application of mannose in enhancing plant root development in oil-contaminated soil, wherein each plant is irrigated with 500 muM of mannose, and irrigation is carried out once every two days, and each time 30 mL of mannose is irrigated. Compared with the prior art, the application discloses that exogenous mannose enhances lateral root development of Mirabilis jalapa and rice, improves the tolerance of rice to oil stress, implies that mannose may play a role as a positive regulator in abiotic stress, and proves that exogenous mannose can participate in root morphological development of plants and has an important role in a plant oil stress recovery pathway; exogenous mannose improves growth and development activity of plants by activating stress resistance related transcription factors and an antioxidant system, and improves the tolerance of the plants to oil stress.

Description

Technical Field

[0001] This invention relates to the field of the effects of exogenous factors on plant growth and development, and in particular to the application of mannose in enhancing root development of plants in petroleum-contaminated soil. Background Technology

[0002] The plant root system is the first organ to sense abiotic stress (YangWang et al., 2013). The elongation of the taproot, the branching of lateral roots (LR), and the direction of the roots determine the root system structure, enabling plants to adapt to changing environmental conditions (Justamante et al., 2019). The ability of plants to develop lateral roots can be used as an important indicator of the strength of their environmental adaptability (Goh et al., 2019).

[0003] The growth and development of lateral root morphology are regulated and influenced by a variety of factors, such as the expression of related genes in the body, the synthesis and transport of hormones, and the influence of the external environment (Cho et al., 2014; John and Alaerts 2005).

[0004] Mannosidase, or β-1,4-D-mannosidase (EC 3.2.1.25), is a key enzyme in the hydrolysis of mannan. It randomly cleaves the β-1,4 glycosidic bonds in the mannan backbone, producing mannose as the hydrolysis product (Wang et al. 2013). Glycosignal can exert its direct effects through osmotic regulation, scavenging reactive oxygen species, and stabilizing biomembranes and biomolecules. Indirectly, it can respond to and adapt to abiotic stress by initiating downstream glycosignal transduction pathways, activating the expression of stress-related genes, and synthesizing plant hormones, osmotic regulators, and antioxidant systems. However, at high concentrations, mannose can be toxic to plants, inhibiting growth and seed germination, reducing proteolytic activity, suppressing the expression of proteasome genes induced by root starvation, and inducing apoptosis (Pego et al., 1999; Stein andhansen, 1999; Brouquisse et al., 2001). Therefore, whether mannose can act as a signaling molecule in response to abiotic stresses (such as oil pollution) requires further investigation. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of mannose in enhancing plant root development in petroleum-contaminated soil.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0007] Application of mannose in enhancing plant root development in petroleum-contaminated soils.

[0008] Furthermore, each plant is watered with 500 μM mannose every two days, 30 mL each time.

[0009] Preferably, the plants include four o'clock flower and rice.

[0010] Compared with existing technologies, this invention reveals that exogenous mannose enhances the growth and development of Mirabilis jalapa and rice, improves rice's tolerance to petroleum stress, and suggests that mannose may play a positive regulatory role in abiotic stress. It also confirms that exogenous mannose can promote lateral root development and increase lateral root density, thereby participating in plant root morphology development and playing an important role in plant recovery pathways to petroleum stress. Furthermore, exogenous mannose can enhance plant growth and development vitality and improve its tolerance to petroleum stress by activating stress-related transcription factors and antioxidant systems. Attached Figure Description

[0011] Figure 1 The effects of different treatments at different times on the phenotype of Mirabilis jalapa seedlings were investigated. Where: A represents phenotype; B represents aboveground fresh weight; C represents underground fresh weight; D represents plant height; E represents root-to-shoot ratio; F represents aboveground water content; G represents underground water content; scale bar: 5cm; CK represents clean soil; OIL represents petroleum stress; OIL+Man represents petroleum stress with external application of mannose; Man represents clean soil with external application of mannose.

[0012] Figure 2 The effects of different treatments at different times on the phenotype of rice seedlings were investigated. Among them: A is phenotype; B is aboveground fresh weight; C is belowground fresh weight; D is plant height; E is root-to-shoot ratio; F is aboveground water content; G is belowground water content; scale bar is 5 cm; CK is clean soil; OIL is petroleum stress; OIL+Man is petroleum stress with external application of mannose; Man is clean soil with external application of mannose.

[0013] Figure 3 The effects of different treatments at different times on some root indicators of Mirabilis jalapa seedlings were investigated. Among them: A is root length; B is average root diameter; C is rooting depth; D is number of root tips; CK is clean soil; OIL is petroleum stress; OIL+Man is petroleum stress with external application of mannose; Man is clean soil with external application of mannose.

[0014] Figure 4 The effects of different treatments at different times on some root indicators of rice seedlings are shown in the figure. Where: A is root length; B is average root diameter; C is rooting depth; D is number of root tips; CK is clean soil; OIL is petroleum stress; OIL+Man is petroleum stress with external application of mannose; Man is clean soil with external application of mannose.

[0015] Figure 5The effects of different treatments at different times on the root surface area and root volume of Mirabilis jalapa seedlings were investigated. Where: A represents root surface area; B represents root volume; CK represents clean soil; OIL represents petroleum stress; OIL+Man represents petroleum stress with external application of mannose; and Man represents clean soil with external application of mannose.

[0016] Figure 6 The effects of different treatments at different times on the root surface area and root volume of rice seedlings were investigated. Where: A represents root surface area; B represents root volume; CK represents clean soil; OIL represents petroleum stress; OIL+Man represents petroleum stress with external application of mannose; and Man represents clean soil with external application of mannose.

[0017] Figure 7 The effects of different treatments on the leaf tissue structure of Mirabilis jalapa over 30 days were shown. A represents the leaf cross-section; B represents the thickness of the upper epidermal cells (TU), palisade tissue (TP), and spongy tissue (TS); C represents the leaf structural looseness (LLS); scale bar 100 μm; * indicates palisade tissue, arrow indicates spongy tissue, triangle indicates upper epidermis, and the same applies below; CK represents clean soil; OIL represents petroleum stress; OIL+Man represents petroleum stress with external application of mannose; Man represents clean soil with external application of mannose.

[0018] Figure 8 The effects of different treatments on the leaf tissue structure of rice over 30 days were investigated. A represents the leaf cross-section; B represents the thickness of the upper epidermal cells (TU), palisade tissue (TP), and spongy tissue (TS); C represents the leaf structure looseness (LLS); scale bar: 50 μm; CK represents clean soil; OIL represents petroleum stress; OIL+Man represents petroleum stress with external application of mannose; Man represents clean soil with external application of mannose.

[0019] Figure 9 The effects of different treatments on the root tissue structure 1 cm below the stem base of Mirabilis jalapa after 30 days; where: A is the root cross section structure; B is the cortex-to-stele ratio (CSR), xylem-to-stele ratio (XSR), and aeren-to-cortex ratio (ACR) of the root tissue structure; scale bar 1000 μm; * indicates endodermis and epidermis, arrow indicates aeren, triangle indicates xylem, the same below; CK is clean soil; OIL is petroleum stress; OIL+Man is petroleum stress with external application of mannose; Man is clean soil with external application of mannose.

[0020] Figure 10The effects of different treatments on the root tissue structure 1 cm below the stem base of rice at 30 days were investigated. Wherein: A is the root cross section structure; B is the cortex-to-stele ratio (CSR), xylem-to-stele ratio (XSR), and aeren-to-cortex ratio (ACR) of the root tissue structure; scale bar 100 μm; CK is clean soil; OIL is petroleum stress; OIL+Man is petroleum stress with external application of mannose; Man is clean soil with external application of mannose.

[0021] Figure 11 The effects of different treatments on the levels of endogenous hormones in the aboveground and underground parts of Mirabilis jalapa and rice over 7 days were investigated. A represents abscisic acid (ABA); B represents salicylic acid (SA); and C represents gibberellin (GA). 3+7 ); CK represents clean soil; OIL represents oil stress; OIL+Man represents oil stress with external application of mannose; Man represents clean soil with external application of mannose.

[0022] Figure 12 The effects of different treatments on root activity of Mirabilis jalapa and rice over 7 days were investigated. CK represented clean soil; OIL represented petroleum stress; OIL+Man represented petroleum stress with mannose application; and Man represented clean soil with mannose application.

[0023] Figure 13 The effects of different treatments on auxin (IAA) in the aboveground and underground parts of Mirabilis jalapa and rice over 7 days were investigated. CK represented clean soil; OIL represented petroleum stress; OIL+Man represented petroleum stress with external application of mannose; and Man represented clean soil with external application of mannose.

[0024] Figure 14 The effects of different treatments at different times on the expression levels of key genes in the IAA response pathway in the underground and aboveground parts of rice were investigated. CK represented clean soil; OIL represented petroleum stress; OIL+Man represented petroleum stress with exfoliated mannose; and Man represented clean soil with exfoliated mannose.

[0025] Figure 15 The effects of different treatments at different times on the expression levels of genes related to root development in the underground part of rice were investigated. CK represented clean soil; OIL represented petroleum stress; OIL+Man represented petroleum stress with exfoliated mannose; and Man represented clean soil with exfoliated mannose.

[0026] Figure 16 The effects of different treatments at different times on the expression levels of stress-related genes in the underground and aboveground parts of rice were investigated. CK represented clean soil; OIL represented petroleum stress; OIL+Man represented petroleum stress with mannose application; and Man represented clean soil with mannose application.

[0027] Figure 17The effects of different treatments at different times on the expression levels of antioxidant-related genes in the underground and aboveground parts of rice were investigated. CK represented clean soil; OIL represented petroleum stress; OIL+Man represented petroleum stress with exfoliated mannose; and Man represented clean soil with exfoliated mannose. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0029] Example 1

[0030] This study investigated the changes in root morphology, plant height, biomass, and water content of plants after exogenous application of mannose under petroleum stress, and analyzed and extracted the principal components of root morphology.

[0031] 1. The rice seeds (Beijing No. 3) used in the experiment were stored in the Biotechnology Laboratory of the College of Biological Science and Technology, Shenyang Agricultural University. The test soil was taken from the experimental base of Shenyang Agricultural University. The petroleum feedstock was provided by PetroChina Liaohe Oilfield and was used to artificially simulate petroleum-contaminated soil. The mannose used in the experiment was purchased from Senyu Technology Co., Ltd.

[0032] 2. The main instrument used in the experiment was a root scanner (EPSON SCANNER).

[0033] 3. Experimental treatment and design.

[0034] (1) The pot experiment was conducted with rice planted in clean soil (CK), artificially simulated petroleum-contaminated soil, artificially simulated petroleum-contaminated soil with mannose applied topically, and clean soil with mannose applied topically. Sampling was performed on days 15, 30, and 45. Each treatment was replicated five times. The initial soil petroleum concentration was 10 g·kg⁻¹. -1 .

[0035] (2) Soil treatment for pot experiments: Clean soil was dug up from designated locations and air-dried naturally in the sun. After removing larger particles, it was sieved through a 4mm sieve. 10g of petroleum was dissolved in 50mL of petroleum ether preheated to 50℃ and mixed with the sieved clean soil to prepare a 10g·kg⁻¹ solution. -1 The contaminated soil continues to dry in the sun, turning it over periodically to ensure the petroleum is evenly distributed. Once the petroleum ether has completely evaporated, thoroughly mix the petroleum-soil mixture and place it in flowerpots to await seedling transplanting.

[0036] (3) Treatment of potted experimental plants.

[0037] a. Seedling Cultivation and Transplanting: Select plump, undamaged, and uniformly sized rice seeds. Soak them in a 5% sodium hypochlorite solution for 10 minutes, then rinse thoroughly with clean water. Place the treated seeds in moist petri dishes (containing two layers of filter paper; place the seeds in the dish, then cover with another layer of filter paper). Germinate in the dark at 28℃. After germination, select seeds of similar growth and transplant them into a 1 / 2 complete nutrient solution for rooting. When the roots reach a length of 2cm or more, transfer the seedlings to pots containing complete nutrient solution. Change the nutrient solution (composition shown in Table 1) every 3 days. When the rice seedlings have grown to the stage of two leaves and one bud, select healthy and uniformly growing seedlings and transplant them into pots containing clean soil and contaminated soil, respectively, with each pot containing 1kg of soil. During the treatment period, maintain a cultivation temperature of 25℃, humidity of 65%, and a light duration of 16 hours followed by 8 hours of alternating light and dark.

[0038] b. Plant treatment: After transplanting, there were 60 pots of plants, 30 of which were in clean soil and the remaining 30 in contaminated soil. The 15 pots in clean soil and the 15 pots in contaminated soil were watered normally, while the rest were watered with 500μM mannose every two days, 30mL each time.

[0039] Table 1 Complete Nutrient Solution Formula

[0040]

[0041] 4. Root sample collection: Plants treated on days 15, 30, and 45 were completely submerged in water. After the soil became loose, the soil on the surface of the roots was gently rinsed off with water. After rinsing, the roots were rinsed with distilled water until there were no impurities (the process was gentle to avoid damaging the roots and affecting the results). The roots were then placed in a glass tank filled with clean water and fully expanded. The roots were scanned to obtain root images for later root index analysis.

[0042] 5. Indicator measurement.

[0043] (1) Root morphology: After scanning the plant roots with a scanner (Espon Scanner), the root images were digitized using a root scanning system (WinRHIZO), and morphological characteristics such as total root length, number of root tips, root surface area, average root diameter and root volume were measured and analyzed.

[0044] (2) Aboveground and belowground biomass: Clean the plant seedlings thoroughly, measure their height and root depth with a ruler, repeating three times; carefully absorb the surface moisture of the plants with filter paper, weigh the aboveground and belowground parts separately to obtain the fresh weight, then place them separately in envelopes, put them in an oven at 105℃ for 15 minutes to kill the green, then dry them at 75℃ to constant weight, and weigh them again to obtain the dry weight, repeating three times. The moisture content and root-to-shoot ratio were measured and calculated using the following formulas: Moisture content = (fresh weight - dry weight) / fresh weight * 100%, Root-to-shoot ratio = belowground fresh weight / aboveground fresh weight.

[0045] 6. Statistical analysis: Experimental data were recorded and statistically analyzed using Excel 2016, and SPSS 23 and GraphPad Prism 8 were used for analysis and plotting.

[0046] Results and Analysis of Example 1

[0047] 1. Aboveground and belowground biomass of plants under different treatments

[0048] like Figure 1 As shown in Figure A, the four o'clocks under different treatments grew well for 15, 30, and 45 days and showed a certain tolerance to oil stress. The four o'clocks under oil stress and those treated with mannose externally had larger root systems than those grown in clean soil. Figure 1 The results showed no significant differences in growth status among the different treatments at 15 days, while the phenotypic trends were consistent at 30 and 45 days. At 30 days, the aboveground fresh weight, plant height, aboveground water content, and underground water content of the four o'clock plant significantly decreased under the OIL treatment, while the OIL+Man treatment showed a significant increase relative to the OIL treatment, reaching the same level as the control (CK). There was no significant difference between the Man and CK treatments. Figure 1 B, D, F, and G); Under OIL treatment, the underground fresh weight was not significantly different from the control (CK), but OIL+Man and Man treatments significantly increased relative to the control (CK). Figure 1 C); The root-to-scap ratio of OIL and OIL+Man was significantly higher than that of CK and Man. Figure 1 E).

[0049] like Figure 2 As shown in Figure A, rice under OIL stress exhibited stunted growth and short root systems at 15, 30, and 45 days of treatment. OIL+Man showed a recovery effect compared to OIL, and the root system was more extensive. The phenotypic trends of the rice treatments were consistent across the three time points. At 30 days of treatment, the aboveground fresh weight, belowground fresh weight, plant height, aboveground water content, and belowground water content of rice under OIL treatment were significantly decreased, while the aboveground fresh weight, plant height, aboveground water content, and belowground water content of rice under OIL+Man treatment were significantly increased compared to OIL treatment. Figure 2 C, D, F, and G); OIL's root-to-crown ratio was higher than that of CK and Man ( Figure 2 E).

[0050] 2. Root length, root diameter, and root absorption area of ​​plants under different treatments

[0051] For four o'clocks ( Figure 3 ), rice Figure 4 Statistical analysis was conducted on total root length, average root diameter, rooting depth, and number of root tips.

[0052] like Figure 3 As shown, after 15 days of treatment, the root length of Mirabilis jalapa treated with OIL and OIL+Man was significantly increased compared to the control (CK). Figure 3 A); the average root diameter of OIL decreased significantly ( Figure 3 B); There was no significant difference in rooting depth among the four treatments. Figure 3 C); OIL+Man significantly increased the number of root apical follicles ( Figure 3 D). After 30 days of treatment, the root length of Mirabilis jalapa was significantly increased compared to the control (CK) under OIL, OIL+Man, and Man treatments, with the increase being extremely significant under Man treatment. Figure 3 A); The average root diameter of OIL+Man increased significantly ( Figure 3 B); There was no significant difference in rooting depth among the four treatments. Figure 3 C); OIL significantly reduced the number of apical buds, while OIL+Man increased the number of apical buds to the same level as CK, and the number of apical buds in Man was significantly higher than that in CK. Figure 3 D). After 45 days of treatment, the root length of Mirabilis jalapa treated with OIL significantly decreased, while the root lengths treated with OIL+Man and Man significantly increased. The root lengths of OIL+Man and CK were similar, while the increase in root length of Man was extremely significant. Figure 3 A); the average root diameter of OIL decreased significantly, while OIL+Man increased significantly compared to OIL and was on par with CK, while Man showed no significant change. Figure 3 B); The rooting depth of the three treatments increased to some extent compared with CK, and the rooting depth of Man increased significantly. Figure 3 C); OIL significantly reduced the number of apical buds, OIL+Man increased the number of apical buds and was on par with CK, and Man significantly increased the number of apical buds (C). Figure 3 D). Overall, the treatment showed some adaptability at 15 days. At 30 and 45 days, OIL treatment significantly inhibited the growth of root length, average root diameter, and number of root tips in Mirabilis jalapa. OIL+Man and Man promoted the growth of root length, average root diameter, rooting depth, and number of root tips.

[0053] like Figure 4As shown, the trends of root length, root depth, and root tip number in rice were consistent across all treatment time points. Taking treatment 45 days as an example, root length, root depth, and root tip number were significantly reduced under the OIL treatment, while OIL+Man treatment significantly increased relative to OIL and was on par with the control (CK). The Man treatment showed a certain degree of increase compared to the CK treatment. Figure 4 (A, C, and D); After 15 days of treatment, the average root diameter of OIL and OIL+Man significantly increased, while Man showed no significant difference compared to CK; After 30 days of treatment, the average root diameter of OIL and Man significantly increased, while OIL+Man significantly decreased compared to OIL and remained the same as CK; After 45 days of treatment, the average root diameter of Man significantly decreased ( Figure 4 B).

[0054] 3. Root surface area and root volume of plants under different treatments

[0055] For four o'clocks ( Figure 5 ), rice Figure 6 Statistical analysis was performed on the root surface area and root volume.

[0056] like Figure 5 As shown, after 15 days of treatment, the root surface area of ​​Mirabilis jalapa (OIL+Man) was significantly increased. Figure 5 A), the root surface area and root volume of Man were significantly reduced ( Figure 5 B). After 30 days of treatment, the root surface area and root volume of *Mirabilis jalapa* (OIL+Man) and Man treatments significantly increased. After 45 days of treatment, the root surface area and root volume of *Mirabilis jalapa* (OIL) treatment significantly decreased. Figure 5 (A, B) OIL+Man significantly increased relative to OIL and was on par with CK, while the root surface area and root volume of Man significantly increased (A, B). Figure 5 A, B).

[0057] like Figure 6 As shown, after 15 days of treatment, the root surface area of ​​rice oil ( Figure 6 A) and root volume ( Figure 6 B) The root surface area of ​​rice with OIL was significantly reduced, while that with OIL+Man was significantly increased relative to OIL. The root surface area and root volume of Man showed no significant changes. After 30 days of treatment, the root surface area of ​​rice with OIL was significantly reduced, while that with OIL+Man was significantly increased. Figure 6 A); OIL+Man and Man root volume increased significantly ( Figure 6 B). After 45 days of treatment, the root surface area and root volume of rice oil were significantly reduced ( Figure 6 (A, B) OIL+Man significantly increased relative to OIL, and Man significantly increased root surface area and root volume. Figure 6 A, B).

[0058] 4. Principal Component Analysis

[0059] The root growth status indicators obtained from the above experiments, including root length, root surface area, root volume, number of root tips, average root diameter, rooting depth, underground biomass, underground water content, and root-shoot ratio, were subjected to correlation analysis. As shown in Table 2, the number of root tips was highly correlated with root length, root surface area, and root volume, and showed a positive correlation; the correlation between root volume and root surface area was the strongest, reaching 0.961; the correlation between underground water content and the other eight indicators was weak, and a negative correlation existed; the remaining indicators all showed some degree of correlation.

[0060] Table 2 Correlation analysis among various phenotypic indicators of plant roots.

[0061]

[0062]

[0063] Note: * indicates a significant difference. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0064] Therefore, eight indicators related to root growth, including root length (X1), root surface area (X2), root volume (X3), number of root tips (X4), average root diameter (X5), rooting depth (X6), underground biomass (X7), and root-to-shoot ratio (X8), were selected for principal component analysis. The suitability of the data was tested using the KMO test and Bartlett's test of sphericity. The results showed that the KMO value was 0.782, indicating a certain correlation among the indicators. The approximate chi-square value of Bartlett's test of sphericity was 593.401, with a significance value of 0.000, indicating that the rejection correlation coefficient of the data matrix was an identity matrix, meaning that the indicators were correlated. Correlation analysis, the KMO test, and the Bartlett's test of sphericity all indicate that the data used is suitable for principal component analysis.

[0065] As shown in Table 3, the first two indicators extracted from the eight indicators explained 76.542% of the total variance, indicating that the first two components, namely root tip number and root surface area, can represent 76.542% of the information of the original eight root growth indicators. The extracted principal components are reliable for evaluating root growth.

[0066] Table 3 Explanation of Total Variance

[0067]

[0068] Note: The extraction method was principal component analysis.

[0069] Based on the two principal component score coefficients (Table 4), we obtain the two linear combinations of Y1 and Y2, i.e., the principal component expressions:

[0070] Y1=0.1784X1+0.1985X2+0.1977X3+0.2013X4+0.1596X5+0.1179X6+0.1477

[0071] X7 + 0.015X8;

[0072] Y2=0.0059X1-0.1414X2-0.1714X3-0.0898X4-0.2358X5+0.4851X6+0.3519

[0073] X7 + 0.4861X8;

[0074] As can be seen from the above formula, in the principal component Y1, the absolute values ​​of the coefficients of root length (X1), root surface area (X2), root volume (X3), number of root tips (X4), and average root diameter (X5) are greater than the absolute values ​​of the coefficients of other variables. Therefore, the principal component Y1 is a comprehensive reflection of the five root growth indicators. It represents the root system configuration of the plant, indicating that these five root growth indicators are essential for examining the plant's growth status.

[0075] In principal component Y2, the absolute values ​​of the coefficients of rooting depth (X6), underground biomass (X7), and root-to-shoot ratio (X8) are greater than the absolute values ​​of the coefficients of other variables. Therefore, principal component Y2 is a comprehensive reflection of these four root growth indicators. It represents the nutritional status of plant roots. Only by rationally allocating resources can plants better complete their entire life cycle.

[0076] Table 4 Component Score Coefficient Matrix

[0077]

[0078] Note: The extraction method was principal component analysis.

[0079] Table 5. Comprehensive Score of Plant Root System Indicators

[0080]

[0081] The comprehensive score expression is derived based on the variance percentages of the two principal components (Table 3):

[0082] Y = 0.5890Y1 + 0.1764Y2;

[0083] The principal component scores and composite scores for the three treatments (CK, OIL, and OIL+Man) were calculated. Table 5 shows that, based on the principal component Y1 score, the OIL treatment score was lower than the CK score, indicating that OIL treatment significantly inhibited root growth. The OIL+Man score was higher than the CK score and significantly higher than the OIL score, indicating that exogenous mannose application had a certain improving effect on root architecture under petroleum stress. Based on the principal component Y2 score, all treatment scores were negative, indicating that plants in all environments tended to allocate nutrients to the aboveground parts. The OIL treatment score was higher than the CK score, indicating that OIL treatment significantly inhibited plant growth, and plants allocated nutrients to root growth to maintain vitality. The OIL+Man score was lower than the CK score and significantly lower than the OIL score, indicating that exogenous mannose application had a certain improving effect on root-shoot nutrient allocation under petroleum stress. Based on the composite score, the OIL treatment score was close to 0, indicating that the soil concentration was 10 g·kg⁻¹. -1 Oil stress has a severe inhibitory effect on plant root development. External application of mannose can improve root development in plants under oil stress, thereby increasing the plant's tolerance to oil stress.

[0084] Example 2

[0085] The changes in root and leaf anatomy under petroleum stress with exogenous mannose were analyzed and compared, and changes in endogenous hormones and root activity were measured.

[0086] 1. Test materials: Same as in Example 1.

[0087] 2. Experimental instruments: The main instruments used in the experiment were an upright microscope (Axio ScopeA1), a rotary evaporator (IKARV8), an LC-MS (LCMS-8050), and an ultra-micro UV spectrophotometer (Malcom ES-2).

[0088] 3. Experimental treatment and design:

[0089] The pot experiment consisted of rice plants grown in four treatments: clean soil (CK), artificially simulated petroleum-contaminated soil, artificially simulated petroleum-contaminated soil with mannose applied topically, and clean soil with mannose applied topically. Sampling was conducted on days 7 and 30. Each treatment was replicated in five places. The initial soil petroleum concentration was 10 g / kg. -1 .

[0090] The soil treatment for the pot experiment was the same as in Example 1.

[0091] The treatment of potted plants was the same as in Example 1.

[0092] 4. Sample collection: The three types of plants treated on day 7 and day 30 were completely submerged in water. After the soil became loose, the soil on the surface of the roots was gently rinsed off with water. After rinsing, the roots were rinsed with distilled water until there were no impurities (the process was gentle to avoid damaging the roots and affecting the results).

[0093] 5. Indicator Measurement

[0094] (1) Paraffin sections:

[0095] Root and leaf samples were taken from plants after 30 days under four treatments, with samples taken from the same location for each material. Root samples were taken 1 cm below the stem base, and young leaf sections were selected. The sampling was repeated three times. Safranin-fast green staining was used; specific experimental procedures are described in (Kang Yunyan, 2022). This experiment employed an upright microscope for examination, and a microscopic imaging system was used for photography and data acquisition. Leaf structural looseness was calculated as spongy tissue thickness / leaf thickness (Liu Hangning et al., 2022).

[0096] (2) Extraction and determination of hormones:

[0097] a. Seedlings under four treatments were sampled at 7 days old, and samples were taken from both above-ground and underground parts. Each sample was weighed 0.5g and repeated 3 times. The samples were then thoroughly ground.

[0098] b. Place the ground sample into a 10mL test tube, add 4mL of extraction solution (methanol: distilled water: concentrated formic acid = 15:4:1), sonicate for 45min, centrifuge at 8000rpm for 5min, and take the supernatant and add it to a new 10mL centrifuge tube.

[0099] c. Repeat step b.

[0100] d. Centrifuge the obtained supernatant at 8000 rpm for 10 min.

[0101] e. Rinse the HLB column three times with 2 mL of methanol, followed by 2 mL of 1 mol·L⁻¹ methanol. -1 Activate the HLB column with formic acid, pass 2 mL of the supernatant obtained in step 4 through the HLB column, and collect the filtrate into a 50 mL rotary evaporator flask.

[0102] f. At 40℃ and 100 rpm, rotary evaporate to 1 mL. Pass the evaporated liquid through a pre-treated MXC column (treatment method is the same as for HLB columns), discard the waste liquid, and use 0.5 mL of 1 mol·L⁻¹ solution. -1 Rinse with formic acid and discard the waste liquid.

[0103] g. Add 2 mL of methanol to the MXC column and collect the filtrate into a clean 50 mL rotary evaporator flask.

[0104] h. Dry the resulting filtrate at 40°C and 100 rpm.

[0105] i. Add 0.5 mL of chromatographic grade methanol to the rotary evaporator flask that was evaporated to dryness in the previous step, and dissolve completely. Filter the resulting solution through a 0.22 μm organic phase filter membrane, collect it in a liquid chromatography vial, and store it at -20 °C for subsequent analysis.

[0106] (3) Root vigor determination: Roots of plant seedlings under four treatments were collected at 7 days. The root vigor test kit (TTC colorimetric method) from Addison Biotechnology Co., Ltd. was used to detect the root vigor of the plants. For detailed operating procedures, please refer to the instruction manual.

[0107] 6. Statistical analysis: The experimental data were statistically analyzed using Excel 2016, and GraphPadPrism 8 was used for analysis and plotting.

[0108] Results and Analysis of Example 2

[0109] 1. Effects of exogenous mannose on the structure of different plant tissues under petroleum stress

[0110] Since both Mirabilis jalapa and rice showed significant phenotypic differences from the control group after 30 days of oil stress, to observe the effects of oil stress and exogenous mannose on the tissue structure of the two plants, cytological methods were used to observe the leaf cross-section structure (perpendicular to the veins) of Mirabilis jalapa and rice treated for 30 days under clean soil (CK), oil stress (OIL), oil stress with exogenous mannose (OIL+Man), and clean soil with exogenous mannose (Man). Figure 7 A and Figure 8 A) and the root cross section 1 cm below the stem base ( Figure 9 A and Figure 10 A). Statistical analysis was performed on the thickness of upper epidermal cells (TU), palisade tissue (TP), spongy tissue (TS), and leaf structure porosity (LLS). Figure 7 B) and Figure 8 B), and statistical analysis was conducted on the radius ratios of root tissue structures: cortex to stele ratio (CSR), xylem to stele ratio (XSR), and aerenchyma to cortex ratio (ACR). Figure 9 B and Figure 10 B).

[0111] like Figure 7As shown in Figure A, the cell walls of Mirabilis jalapa leaves were green under all treatments. The epidermal cells, palisade tissue, and spongy tissue of Mirabilis jalapa leaves grown in the CK and Man environments were tightly packed and clearly visible. After OIL treatment, leaf cells were damaged, the palisade tissue was irregularly arranged, and the spongy tissue cells showed signs of breakage. Leaves treated with OIL+Man showed a more regular arrangement of palisade tissue compared to those treated with OIL, and no breakage of spongy cells was observed. Statistical results showed that OIL significantly reduced TU and TP in Mirabilis jalapa leaves, while significantly increasing TS and LLS. Under OIL+Man treatment, TP significantly increased, TS decreased compared to OIL treatment but significantly increased relative to CK, and LLS was the same as OIL but significantly increased compared to CK. Under Man treatment, TS and LLS significantly increased compared to CK. Figure 7 (B and C).

[0112] like Figure 8 As shown in Figure A, the cell walls of rice leaves were green under all four treatments. In the CK and Man environments, the upper and lower epidermal cells, palisade tissue, and spongy tissue of rice leaves were tightly packed and clearly visible. After OIL treatment, the palisade tissue could not be clearly observed, the intercellular spaces of the spongy tissue increased, and fragmentation was observed. In the OIL+Man treatment, compared to OIL, the palisade tissue of the leaves was clearly and regularly arranged, and no fragmentation was observed in the spongy tissue, which was also tightly packed. Statistical results showed that OIL significantly reduced TU, TP, TS, and LLS in rice leaves; under the OIL+Man treatment, TS and LLS significantly increased compared to the OIL treatment, while TU and TP remained the same as those under OIL; under the Man treatment, TP and TS significantly increased compared to the CK treatment. Figure 8 (B and C).

[0113] like Figure 9 As shown in Figure A, the epidermal cells of the root 1 cm below the stem base of *Mirabilis jalapa* significantly thickened under OIL treatment, exhibiting tissue rupture and an increased stele area. Compared to OIL treatment, OIL+Man treatment resulted in thinner epidermal cells with intact structure and denser cell arrangement. Both CK and Man treatments showed intact structure and neat, dense cell arrangement. The root diameter of OIL+Man and Man treatments significantly increased. Statistical results indicate that OIL treatment significantly reduced the xylem-to-stele ratio (XSR) and aerenchyma-to-cortex ratio (ACR) in *Mirabilis jalapa* root tissues. OIL+Man treatment significantly increased XSR and ACR compared to OIL treatment, while the cortex-to-stele ratio (CSR) significantly decreased. Man treatment significantly reduced both CSR and XSR in root tissues. Figure 9 B).

[0114] like Figure 10As shown in Figure A, the epidermal cells of the rice root 1 cm below the stem base significantly thickened under OIL treatment, and tissue rupture occurred. In contrast, the OIL+Man treatment showed intact tissue structure, and no rupture was observed. The CK and Man treatments also showed intact structure and orderly cell arrangement. Statistical results indicate that the cortex-to-stele ratio (CSR) of rice root tissues significantly increased after OIL treatment, while the xylem-to-stele ratio (XSR) and the aeren-to-cortex ratio (ACR) significantly decreased. After OIL+Man treatment, CSR significantly decreased and XSR significantly increased compared to OIL treatment. Compared to CK treatment, Man treatment significantly decreased CSR and XSR and significantly increased ACR. Figure 10 B).

[0115] 2. Effects of exogenous mannose application on the content of endogenous hormones in different plants under petroleum stress

[0116] Since hormones are related to plant stress resistance and growth and development, to verify the changes in endogenous hormones after OIL, OIL+Man, and Man treatments, the levels of ABA, SA, and GA in the aboveground and underground parts of Mirabilis jalapa and rice were measured on day 7 of treatment. 3+7 content ( Figure 11 Data shows that OIL treatment affects ABA, SA, and GA. 3+7 Both plants showed an upward trend in both their above-ground and below-ground parts. For example... Figure 11 As shown in Figure A, compared with OIL, the ABA content in both aboveground and belowground parts of the two plants decreased after OIL+Man treatment; compared with CK, there was no significant difference in the aboveground and belowground parts of the two plants after Man treatment. Compared with OIL, the SA content in the belowground part of Mirabilis jalapa increased slightly after OIL+Man treatment, while the content in other tissues decreased; compared with CK, the SA content in the aboveground and belowground parts of Mirabilis jalapa and the aboveground part of rice increased after Man treatment. Figure 11 B) Compared with OIL, OIL+Man treatment resulted in better underground GA in Mirabilis jalapa. 3+7 The content increased, while it decreased in other tissues; compared with the control (CK), the content of Man treatment showed an increasing trend in the underground tissues of both plants. Figure 11 C).

[0117] 3. Effects of exogenous application of mannose on root activity of different plants under petroleum stress

[0118] Root systems of two plants, Mirabilis jalapa and rice, were treated for 7 days with clean soil (CK), petroleum stress (OIL), petroleum stress with mannose (OIL+Man), and clean soil with mannose (Man). Root activity was measured using the TTC method. Results showed that after 7 days of OIL treatment, root activity in Mirabilis jalapa did not change significantly, while root activity in rice decreased significantly. Compared with OIL treatment, the combined treatment of petroleum stress and mannose significantly increased root activity in all three plants. After Man treatment, root activity in all three plants did not change significantly compared to CK. Figure 12 ).

[0119] Example 3

[0120] The changes in the expression levels of genes related to stress resistance, antioxidant activity, auxin signaling pathway, and root development under petroleum stress were determined.

[0121] 1. The test materials are the same as in Example 1.

[0122] 2. Experimental instruments: The main instruments used in the experiment were rotary evaporator (IKA RV8), LC-MS (LCMS-8050), ultra-micro UV spectrophotometer (Malcom ES-2), and Mona q225 real-time PCR instrument.

[0123] 3. Experimental treatment and design:

[0124] (1) Auxin determination: A pot experiment was conducted on rice plants grown in four different treatments: clean soil (CK), artificially simulated petroleum-contaminated soil, artificially simulated petroleum-contaminated soil with mannose application, and clean soil with mannose application. Sampling was performed on day 7. Five replicates were performed for each treatment. The initial soil petroleum concentration was 10 g / kg. -1 .

[0125] (2) qRT-PCR: Pot experiments were conducted on rice plants grown in four different soil types: clean soil (CK), artificially simulated petroleum-contaminated soil, artificially simulated petroleum-contaminated soil with mannose application, and clean soil with mannose application. Sampling times were 12h, 24h, 48h, and 72h. Each treatment was replicated five times. The initial soil petroleum concentration was 10 g·kg⁻¹. -1 .

[0126] (3) Soil treatment for pot experiments:

[0127] a. Treatment of the soil used in the experiment: Same as in Example 1.

[0128] b. Seedling transplanting: Select healthy seedlings that are growing uniformly in clean soil and transplant them separately into flowerpots containing clean soil and contaminated soil, with each flowerpot containing 1 kg of soil. During the transplanting period, maintain a cultivation temperature of 25℃, humidity of 65%, and 16 hours of light and 8 hours of darkness.

[0129] c. Plant treatment: Same as in Example 1.

[0130] 4. Sample collection:

[0131] (1) Auxin determination: The plants were completely submerged in water on the 7th day of treatment. After the soil became loose, the soil on the surface of the roots was gently rinsed off with water. After rinsing, the roots were rinsed with distilled water until there were no impurities (the process was gentle to avoid damaging the roots and affecting the results).

[0132] (2) qRT-PCR: Rice treated at 12h, 24h, 48h and 72h were used, and the remaining operations were the same as those for auxin determination.

[0133] 5. Index Measurement:

[0134] (1) The extraction and determination of auxin are the same as in Example 2.

[0135] (2) RNA extraction and cDNA synthesis: Rice roots and leaves were sampled at 12h, 24h, 48h, and 72h under four different treatments. Three tubes were extracted from each material, with two replicates. Total RNA was extracted from the samples using the Plant Total RNA Extraction Kit from Tiangen Biotech Co., Ltd. The FastKing cDNA First-Strand Synthesis Kit from Mona Co., Ltd. was used. Detailed operating procedures are provided in the instruction manual.

[0136] (3) Real-time quantitative PCR: Using the obtained cDNA as the original template, specific primers for the target gene were designed using Primer 5.0. The rice primer sequences are detailed in Table 6. The primers were synthesized by Suzhou Hongxun Biotechnology Co., Ltd. qRT-PCR was performed using the ChemoHS qPCRMix kit from Mona Biotechnology Co., Ltd. on a Mona q225 real-time quantitative PCR instrument. The qRT-PCR reaction system and reaction procedure are shown in Tables 7 and 8.

[0137] Table 6 Primers for rice qRT-PCR reaction

[0138]

[0139]

[0140] Table 7 qRT-PCR reaction system

[0141]

[0142] Table 8 qRT-PCR reaction procedure

[0143]

[0144] 6. Statistical analysis: The experimental data were statistically calculated using Excel 2016 for each Ct value, and analyzed and plotted using GraphPadPrism 8.

[0145] Results and Analysis of Example 3

[0146] 1. Effects of exogenous application of mannose on the content of different plant growth hormones under petroleum stress

[0147] Since auxin is related to plant growth and development, to verify the changes in endogenous auxin after OIL, OIL+Man, and Man treatments, the IAA content in the aboveground and underground parts of the three plants was measured on day 7. Figure 13 Data showed that IAA levels in both the aboveground and underground parts of the three plant species increased under OIL treatment. Compared with OIL, the IAA content in both the aboveground and underground parts of the three plant species increased after OIL+Man treatment, with the underground part of rice showing the largest increase of 1.54 times. Compared with the control (CK), the IAA content in both the aboveground and underground parts of the three plant species increased to some extent after Man treatment.

[0148] 2. Expression patterns of key genes in the rice IAA response pathway

[0149] Taking rice as an example, the expression of key genes TIR1, AUX1, ARF7, and ARF19 in the underground and aboveground parts of rice under clean soil (CK), oil stress (OIL), oil stress with exogenous mannose (OIL+Man), and clean soil with exogenous mannose (Man) conditions for 12h, 24h, 48h, and 72h of cultivation were detected. The results are as follows: Figure 14 As shown.

[0150] Compared with OIL treatment, OIL+Man treatment mainly upregulated the expression levels of TIR1 in both the aboveground and underground regions; OIL+Man treatment upregulated the expression level of AUX1 in the underground region, while the expression level of the aboveground region was slightly downregulated at 24h and 72h; ARF7 showed a 6.72-fold upregulation in the underground region at 48h, and upregulation in the aboveground region from 12h to 48h; ARF19 showed a significant upregulation in the underground region at 48h and 72h, and upregulation in the aboveground region at 48h and 72h, with a slight downregulation at 24h. Compared with CK, Man treatment significantly upregulated the expression level of TIR1 in the underground region from 24h to 48h, with an overall upregulation trend in the aboveground region; AUX1 showed a mainly upregulation trend in both the underground and aboveground regions; ARF7 showed upregulation in the underground region at 12h and 48h, with no significant changes in expression levels at other time points, while the expression level of the aboveground region was upregulated at all detection time points; ARF19 showed some degree of upregulation in both the aboveground and underground regions.

[0151] 3. Expression patterns of genes related to rice root development

[0152] The expression levels of root development-related genes XTH1, GNOM, CYP2, WOX11, ORC3, and DRO1 in the underground parts of rice were detected after 12 h, 24 h, 48 h, and 72 h of cultivation under clean soil (CK), oil stress (OIL), oil stress with mannose exfoliation (OIL+Man), and clean soil with mannose exfoliation (Man). The results are as follows: Figure 15 As shown.

[0153] Compared with OIL treatment, XTH1 expression was upregulated under OIL+Man treatment, with upregulation of 11.24-fold and 3.75-fold at 48h and 72h, respectively; GNOM and CYP2 showed overall upregulation; WOX11 showed upregulation at 12h and 48h, with no significant changes at other time points; ORC3 showed overall upregulation, with the largest change of 3.85-fold at 24h; DRO1 showed significant upregulation at 12h and 48h, with an upregulation of 2.28-fold at 12h; compared with CK, all root development-related genes detected under Man treatment showed upregulation.

[0154] 4. Expression patterns of stress-resistance-related genes in rice

[0155] The expression levels of stress-resistance-related genes bZIP23, ERF48, WRKY97, NAC2, and PIP2 were detected in rice after 12 h, 24 h, 48 h, and 72 h of cultivation under clean soil (CK), oil stress (OIL), oil stress with exfoliated mannose (OIL+Man), and clean soil with exfoliated mannose (Man) conditions. The results are as follows: Figure 16 As shown.

[0156] In general, the expression of each gene differs between the underground and aboveground parts. Compared with OIL treatment, the expression levels of bZIP23 in both the underground and aboveground parts were downregulated under OIL+Man treatment, with a highly significant downregulation at 72h; the expression level of ERF48 in the underground part was upregulated, with a highly significant upregulation at 48h; the expression level of WRKY97 in the underground part was significantly upregulated, with the most significant change at 12h, while the expression level in the aboveground part was downregulated at 12h and upregulated at 24h; the expression level of NAC2 in the underground part was continuously upregulated at 48h and 72h, while the expression level in the aboveground part mainly showed a downregulation trend, with the most significant change at 72h; PIP2;3 showed upregulated expression at 24h and 48h, and downregulated expression at 72h. Compared with CK, under Man treatment, the expression of bZIP23 underground fraction was upregulated at all detection time points, while the expression of aboveground fraction was downregulated at all detection time points; ERF48 underground fraction expression was significantly upregulated at 48h and 72h, and aboveground fraction expression was upregulated at 24h; WRKY97 underground fraction expression was upregulated at 24h and 72h, with no change at other time points, while aboveground fraction expression was significantly upregulated at 24h and significantly downregulated at 12h and 72h; NAC2 underground fraction mainly showed an upregulated trend, while aboveground fraction expression was not significantly changed except at 12h; PIP2;3 underground fraction expression was upregulated at 24h and 48h, with no significant change at other time points, while PIP2;3 aboveground fraction expression was upregulated at 48h, with no significant change at other time points.

[0157] 5. Expression patterns of antioxidant-related genes in rice

[0158] The expression levels of antioxidant-related genes SODCC.1, POD2, CATB, APX2, and GSTU6 in rice were detected after 12 h, 24 h, 48 h, and 72 h of cultivation under clean soil (CK), oil stress (OIL), oil stress with exfoliated mannose (OIL+Man), and clean soil with exfoliated mannose (Man) conditions. The results are as follows: Figure 17 As shown.

[0159] Compared with OIL treatment, OIL+Man treatment significantly upregulated the expression levels of SODCC.1 in the underground fraction at 24h and 48h, and upregulated at 72h. The expression levels of the aboveground fraction were also significantly upregulated at 24h, 48h, and 72h. For POD2, the underground fraction was upregulated at 12h and 72h, and significantly downregulated at 24h, while the aboveground fraction showed a downward trend. For CATB, the underground fraction was significantly upregulated at all time points, with a 1.39-fold increase at 72h. The aboveground fraction was upregulated at 48h and 72h, with no significant changes at other time points. For APX2, the underground fraction showed an upward trend, while the aboveground fraction remained largely unchanged, with only a slight upregulation at 24h. For GSTU6, the underground fraction was upregulated, the aboveground fraction was slightly downregulated at 24h, and significantly upregulated at 48h and 72h. Compared with CK, SODCC.1 expression was significantly upregulated in the underground part under Man treatment, and the expression level in the aboveground part was significantly upregulated at 24h and 48h. POD2 expression mainly showed an upregulated trend in the underground part, and the expression level in the aboveground part was upregulated at 24h, with no significant changes at other time points. CATB expression was upregulated in the underground part at all time points except for 12h, and the expression level in the aboveground part was also upregulated. APX2 expression was upregulated in the underground part, with no significant difference in the aboveground part. GSTU6 expression increased significantly in the underground part at 12h and 24h, and the expression level in the aboveground part was upregulated from 24h to 72h.

[0160] In summary, exogenous mannose enhanced the growth and development of rice and improved its tolerance to petroleum stress, suggesting that mannose may play a positive regulatory role in abiotic stress. It also confirmed that exogenous mannose can participate in root morphology development and plays an important role in the recovery pathway of plant tolerance to petroleum stress. qRT-PCR identification results in rice showed that exogenous mannose improves plant growth and development vitality and enhances its tolerance to petroleum stress by activating stress-related transcription factors and antioxidant systems.

[0161] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. The application of mannose in enhancing plant root development in petroleum-contaminated soil, characterized by: Each plant was irrigated with 500 μM mannose every two days, 30 mL each time; the plants were Mirabilis jalapa or rice; the mannose promoted lateral root development and increased root tip number by regulating the expression of auxin signaling pathway-related genes AUX1, ARF7, ARF19 and / or key root development genes WOX11, DRO1; the mannose improved the plant's tolerance to petroleum stress by activating the expression of stress-resistance-related genes bZIP23, NAC2 and / or antioxidant-related genes SODCC.1, CATB; root development included lateral root development, increased root tip number, and increased root surface area and volume; the petroleum concentration in the contaminated soil was 10 g·kg⁻¹. -1 .

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