Application of nanomimetic PMO in regulating rice leaf color traits
Patent Information
- Application Number
- CN202610917940.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-24
- Publication Date
- 2026-08-21
AI Technical Summary
[0018] This invention uses nano-enzyme mimics PMO to regulate rice leaf color mutants, revealing their photosynthetic pigment metabolism regulation and stress resistance mechanism. It provides a reference for the application of nanomaterials in agriculture and also provides nanotechnology and application pathways for stress regulation in rice seedling transplanting management.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of nanoenzyme technology, specifically relating to the application of nanoenzyme PMO (manganese tetroxide nanoparticles) in regulating the leaf color trait of rice. Background Technology
[0002] Rice (Oryza sativa L.) is one of the world's major food crops, and its yield and quality are directly related to food security and sustainable agricultural development. During rice growth, leaves, as the main organs of photosynthesis, bear the core functions of capturing light energy, fixing carbon dioxide, and synthesizing organic matter. Their physiological state directly determines the plant's growth rate, nutrient accumulation, and final yield. Leaf health is closely related to photosynthetic efficiency, and the normal development of leaves and chloroplasts during the seedling stage is particularly crucial, directly affecting nutrient accumulation and plant resistance.
[0003] In studies of rice leaf physiology and chloroplast development, leaf color mutations provide important experimental materials for revealing chlorophyll biosynthesis, photosynthetic pigment ratios, and metabolic regulation. Leaf color mutations in seedlings mainly manifest as yellowing, whitening, striping, or green restoration. These mutations not only affect early growth but also reflect photosynthetic efficiency and the plant's response to environmental stress. The occurrence of leaf color mutations is usually accompanied by decreased chlorophyll content, changes in carotenoid ratios, accumulation of reactive oxygen species (ROS), and abnormal sugar metabolism. These changes further affect photosynthetic capacity and plant homeostasis. Leaf color mutations are not merely changes in appearance; they are a comprehensive reflection of plant photosynthetic metabolism, antioxidant capacity, and hormone signaling regulation.
[0004] The mutation of rice seedling leaf color and its regulatory mechanism have significant scientific importance and wide application value. Methods for regulating rice leaf color traits are urgently needed in this field. Summary of the Invention
[0005] The purpose of this invention is to provide the application of nano-enzyme mimic PMO (manganese tetroxide nanoparticles) in regulating the leaf color trait of rice. The PMO described in this invention can effectively alleviate leaf sugar deficiency and regulate sugar distribution and reactive oxygen species balance.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] This invention provides the application of nano-enzyme PMO in regulating the leaf color trait of rice, wherein the nano-enzyme PMO is an SOD-type polyacrylic acid-manganese oxide nano-enzyme.
[0008] Preferably, the preparation method of the SOD-type polyacrylic acid-manganese oxide nano-enzyme is as follows: after mixing the manganese sulfate aqueous solution and the polyacrylic acid aqueous solution evenly, the mixture is added dropwise to the ammonium hydroxide solution and stirred; the stirred solution is reacted in an oven, the supernatant is obtained by centrifugation and then dialyzed, and the dialyzed solution is dried to obtain the SOD-type polyacrylic acid-manganese oxide nano-enzyme.
[0009] Preferably, the concentration of the nano-enzyme PMO is 200 mg / L.
[0010] This invention also provides the application of the above-mentioned nano-enzyme PMO in promoting plant growth.
[0011] Preferably, promoting plant growth means increasing plant height and leaf area.
[0012] Preferably, promoting plant growth involves increasing chlorophyll content.
[0013] This invention also provides the application of the above-mentioned nano-enzyme PMO in regulating sugar distribution and reactive oxygen species balance in rice zebra leaves.
[0014] This invention also provides the application of the above-mentioned nano-enzyme PMO in regulating sucrose metabolism and transport-related genes and trehalose metabolism genes.
[0015] Preferably, the sucrose metabolism and transport-related genes are: SPS, SPP, and SUT2; and the trehalose metabolism genes are TPS2 and TPP1.
[0016] This invention also provides the application of the above-mentioned nano-enzyme PMO in improving the SPAD value of rice leaves.
[0017] Beneficial effects:
[0018] This invention uses nano-enzyme mimics PMO to regulate rice leaf color mutants, revealing their photosynthetic pigment metabolism regulation and stress resistance mechanism. It provides a reference for the application of nanomaterials in agriculture and also provides nanotechnology and application pathways for stress regulation in rice seedling transplanting management.
[0019] In this invention, PMO significantly alleviates zebra leaf phenotype and increases photosynthetic pigment content, particularly chlorophyll a, b, and total chlorophyll content in the yellow-green stripes, while simultaneously reducing the chlorosis rate and demonstrating a significant regreening effect. PMO effectively alleviates sugar deficiency, regulates sugar distribution and reactive oxygen species balance, and promotes leaf regreening. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 Phenotypic characteristics of deeply transplanted zebra leaves under different spraying treatments.
[0022] Figure 2 Analysis of chlorophyll content in zebra leaves with yellow-green stripes under different treatment conditions.
[0023] Figure 3 Analysis of sucrose, hydrogen peroxide, and superoxide anion content in the yellow-green stripes of zebra leaves.
[0024] Figure 4 This study analyzed the expression of genes involved in sugar metabolism, antioxidant enzymes, ABA synthesis, and chlorophyll synthesis in zebra leaves. The left side of the upper left shows transcriptome analysis, the right side shows quantitative fluorescence verification, and the bottom side shows quantitative fluorescence detection analysis of genes.
[0025] Figure 5 Phenotypic diagram of PMO spraying treatment during the seedling stage.
[0026] Figure 6 Phenotypic diagram of sword leaves treated with PMO spraying.
[0027] Figure 7 Analysis of photosynthetic pigments in sword leaves treated with PMO spraying.
[0028] Figure 8 Analysis of the surface area of sword leaves treated with PMO spraying.
[0029] Figure 9 Transcriptome analysis of gene expression related to photosynthesis, chlorophyll synthesis and sugar metabolism in red rice flag leaves under PMO spraying treatment. Detailed Implementation
[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0031] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.
[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.
[0033] Nanomaterials: Preparation of SOD-like poly(acrylic) acid coated Mn3O4 nanoparticles (PMO): 0.425 g of manganese sulfate and 4.500 g of polyacrylic acid (PAA, 1800MW, Sigma Aldrich) were weighed and dissolved in 2.5 mL and 5 mL of pure water, respectively. The two solutions were vortexed at 2000 r / min for 15 min to ensure thorough mixing. The mixture was then added dropwise to a 30 mL brown bottle containing 15 mL of ammonium hydroxide solution (30%, Sigma Aldrich). The mixture was then stirred on a magnetic stirrer at room temperature for 24 h. The solution was then transferred to a reaction vessel and placed in an oven at 120 °C for 24 h. The solution was then removed from the reaction vessel and aliquoted into 2 mL centrifuge tubes, centrifuged at 4000 r / min for 1 h to precipitate large particles. The supernatant was collected and transferred to a dialysis bag (MW10 K, Millipore) for dialysis for 24 h. The dialysis solution was then transferred to a foil dish dried to constant weight, and its weight before drying was accurately measured using a balance. The solution was then placed in an oven (80 °C) and dried until the weight was constant, and its weight was accurately measured again. The concentration of PMO was calculated based on the original weight of the sample and the weight lost after drying. Finally, water was added to prepare a 200 mg / L aqueous solution (the optimal spraying concentration determined by previous concentration screening by our research group), which was then stored at 4 °C for later use.
[0034] Example 1: Effects and physiological mechanisms of PMO on leaf color regulation in rice seedlings
[0035] Rice material: A leaf color mutant B03F obtained from the progeny of the photoperiod-thermosensitive male sterile line Efeng 1S. After deep transplanting, the leaf sheath and leaf blade exhibited transverse discontinuous chlorosis perpendicular to the leaf vein direction (i.e., zebra leaf).
[0036] Seedlings at the 3-4 leaf stage were used for both deep and shallow transplanting treatments. The transplanting depth was set at 8 cm and 3 cm above the leaf sheath, respectively. Deeply transplanted plants exhibited zebra-leaf phenotype, while shallowly transplanted plants did not. The zebra-leaf phenotype appeared 4-6 days after transplanting and occurred on the new leaves.
[0037] The specific application time was scheduled for the day after transplanting, using 200 mg / L PMO, 26.4 mg / L ABA (Yuanye, Shanghai, China), and distilled water respectively. All solutions were supplemented with 0.5% (v / v) Silwet L-77 surfactant (Yuanye, Shanghai, China). Spraying was done at 6 PM, covering the above-ground parts of the plant, until the leaves were moist and began to drip. Spraying was repeated once daily for a total of 5 times, until the zebra leaf characteristics were significantly restored.
[0038] The specific processing group details are as follows:
[0039] The group that was shallowly transplanted and sprayed with H2O had new leaves that were normally green and served as the control group (CK).
[0040] Deep transplanting and H2O spraying resulted in zebra leaves exhibiting green and yellow stripes (HG, HY).
[0041] In the deep transplanting and PMO spraying group, zebra leaves showed green and yellow stripes (PG, PY).
[0042] The deep transplanting and ABA spraying group served as a parallel control group for the occurrence and recovery of zebra leaf phenotype.
[0043] (1) Observe and record the leaf phenotypes of rice in each treatment. The results are as follows: Figure 1 As shown. Shallow transplanting + H2O treatment (CK) resulted in normal dark green leaves without any chlorotic streaks. Deep transplanting + H2O treatment resulted in distinct transverse discontinuous chlorotic streaks, a typical zebra leaf phenotype; HG represented green streaks, and HY represented yellow streaks, with bright yellow areas and clear boundaries between the yellow and green areas. Deep transplanting + PMO and deep transplanting + ABA treatments, while still showing some streaks, resulted in significantly greener yellow streaks, a significantly reduced number of streaks, and an expanded green area, demonstrating significant overall regreening / greening effects. Compared to deep transplanting + H2O treatment, the zebra leaf symptoms were significantly reduced in the PMO and ABA treatment groups. However, it was also clearly observed that rice growth slowed after ABA spraying; ABA is a stress hormone that achieves its stress-resistance effect by slowing plant growth. In contrast, PMO spraying accelerated plant growth, resulting in significantly increased plant height, vigorous growth, and larger leaf area, promoting plant growth. The proportion of yellow-green area in leaves for each treatment was statistically analyzed using OpenCV software. The results are as follows: Figure 1 As shown.
[0044] The results of chlorophyll content measurement of leaves under each treatment are as follows: Figure 2As shown in the figure. Compared with the control (CK), the chlorophyll a, chlorophyll b, and total chlorophyll contents of the deep transplanting + H2O treatments (HG, HY) were significantly reduced (P<0.05), and the reduction was greater in HY than in HG. After deep transplanting + PMO treatment, the chlorophyll a, chlorophyll b, and total chlorophyll contents of PG and PY were significantly higher than those of the corresponding deep transplanting + H2O treatments (P<0.05).
[0045] The above results indicate that PMO significantly alleviates zebra leaf phenotype and increases photosynthetic pigment content, especially chlorophyll a, b and total chlorophyll content in yellow-green stripes, while reducing the chlorosis rate and showing a significant regreening effect, which is better than ABA.
[0046] (2) Analysis of sucrose, hydrogen peroxide and superoxide anion content in the yellow-green stripes of zebra leaves, the results are as follows: Figure 3 As shown, deep transplanting induces the zebra-leaf phenotype in rice leaves, which physiologically manifests as oxidative stress and sugar deficiency, particularly in the yellow stripes of the zebra leaves, where superoxide anion content increases while sucrose content decreases significantly. After PMO treatment, sucrose content in the yellow-green stripes of the zebra leaves significantly increases, and superoxide anion content decreases. This indicates that PMO effectively alleviates leaf sugar deficiency and oxidative stress caused by deep transplanting, regulates sucrose distribution and reactive oxygen species balance, and promotes zebra leaf regreening.
[0047] (3) Analysis of gene expression for sugar metabolism, antioxidant enzymes, ABA synthesis, and chlorophyll synthesis in zebra leaves. The results are as follows: Figure 4 As shown, under deep transplanting conditions, PMO treatment upregulated the expression of the chlorophyll b synthase gene CHYB and the antioxidant gene APX, indicating a gradual recovery in chlorophyll synthesis. Simultaneously, sucrose metabolism and transport-related genes SPS, SPP, and SUT2, as well as trehalose metabolism genes TPS2 and TPP1, were significantly upregulated, confirming that PMO effectively alleviated sugar deficiency, coordinated sugar distribution, and promoted leaf regreening in zebra plants.
[0048] Example 2: The universality of PMO in regulating leaf color during the seedling stage
[0049] Rice materials: Red rice and 9311.
[0050] The study used two rice varieties, Luotian Red Rice and Yangdao No. 6, which were preserved in the laboratory and their specific sources are as follows.
[0051] The high-quality specialty colored rice variety "Luotian Red Rice", abbreviated as RR, is a special red rice variety developed by the research team through the improvement and breeding of local high-quality varieties.
[0052] The high-quality conventional rice variety "Yangdao 6", abbreviated as 9311, has typical indica rice genetic characteristics. It has good yield potential and temperature resistance, and is often used as a restorer line in three-line hybrid rice breeding. Several high-yielding hybrid combinations have been bred from it.
[0053] Nanoscale regulation measures: Foliar spraying of 200 mg / L PMO for regulation.
[0054] This embodiment analyzes the effects of foliar spraying of nano-enzyme PMO on the growth of rice seedlings.
[0055] Seedlings at the 3-leaf stage were transplanted, and sprayed with 200 mg / L PMO the day after transplanting. 0.5% (v / v) Silwet L-77 surfactant (Yuan Ye, Shanghai, China) was added to the above solution. Spraying was performed at 6 PM, covering the above-ground parts of the plants until the leaves were moist and began to drip. This was repeated once.
[0056] Samples were taken on the seventh day after the spraying treatment to measure the leaf surface area and SPAD value.
[0057] The results are as follows Figure 5 As shown, under natural light and temperature conditions, spraying with 200 mg / L PMO significantly promoted the increase of SPAD value in seedling leaves without inhibiting or toxicizing their growth, demonstrating good biocompatibility. The SPAD value of leaves in the PMO-sprayed group increased by 18.7%, and the plant height and aboveground dry weight increased by 12.3% and 21.5%, respectively.
[0058] Example 3: The effect of PMO on regulating the color of sword-shaped leaves and its universality
[0059] Rice materials: Red rice and 9311
[0060] The materials are as described in Example 2.
[0061] Nanoscale regulation measures: Foliar spraying of 200 mg / L PMO for regulation.
[0062] Rice cultivation experiments were conducted indoors using a potted planter. Seed soaking and germination treatment were carried out on May 6th, and sowing was completed on May 11th, with seedlings cultivated in pots. On June 10th, seedlings with uniform growth and robust development were selected and transplanted into experimental pots. Throughout the growing season, water and fertilizer were supplied uniformly, and routine pest, disease, and weed control was implemented. The growth dynamics and performance of the rice plants were regularly observed and recorded during the experiment.
[0063] Spraying treatment and sampling methods
[0064] The reproductive growth stage experiment involved two foliar sprays of nano-enzyme. Specifically, the first spray was applied during the third stage of spikelet differentiation, and the second spray was applied on the day of heading. The spray solution was 200 mg / L PMO (with 0.5% (v / v) Silwet L-77 surfactant added). The spraying time was 6 pm, covering the above-ground parts of the plant until the leaves were moist and began to drip.
[0065] Samples were taken after the second spraying treatment to measure the surface area and SPAD value of the sword leaves, the content of photosynthetic pigments, the content of reactive oxygen species, and to perform transcriptome analysis.
[0066] Analysis of SPAD value and surface area of sword leaves
[0067] The SPAD values of rice leaves for each treatment were measured using a SPAD-502 chlorophyll meter. Ten representative plants for each treatment were selected and measured on the same morning. Measurements were taken at evenly spaced points on each leaf, avoiding the main vein. Measurements were taken three times at the middle and upper middle parts of the leaf, and the average value was recorded.
[0068] Surface area analysis was performed as follows: Five representative plants were selected for each treatment, and complete leaves were collected and immediately scanned at 300 dpi resolution using an Epson Perfection V850 Pro scanner to obtain leaf images. ImageJ software (National Institutes of Health, USA) was used to analyze the scanned images and calculate the leaf surface area (cm²). Leaf length and maximum width were measured, and the leaf area index was calculated.
[0069] Detection of reactive oxygen species content in sword leaves
[0070] The hydrogen peroxide and superoxide anion contents in leaves were determined using a hydrogen peroxide test kit (visible spectrophotometry, Keming H2O2-2-Y) and a superoxide anion content test kit (micro-method, Keming SA-2-G).
[0071] Detection of photosynthetic pigment content in sword leaves
[0072] The assay was performed using a kit (Yuanye Biotechnology Co., Ltd., Shanghai, China). First, 0.1 g of fresh sample was taken, and 50 mg of extraction powder and 1 mL of Carotenoid Assay Buffer were added. The mixture was ground in an ice bath and transferred to a 10 mL centrifuge tube. The mortar was rinsed with a small amount of buffer, and the liquids were combined and the buffer was added to a final volume of 10 mL. The mixture was then incubated in the dark for 5 min–2 h. Next, the sample was centrifuged at 4000 r / min for 5 min, and the supernatant was collected for analysis. The spectrophotometer was preheated for 30 min and adjusted to wavelengths of 665 nm, 649 nm, and 470 nm. Zeroing the sample with the buffer, the absorbance of the extract at the corresponding wavelengths was measured (A665, A649, A470).
[0073] The results are as follows Figure 6-8 As shown, after PMO treatment, the sword-shaped leaves of both Red Rice and 9311 turned dark green, with well-developed leaves and a significantly increased leaf area, and the effect was more pronounced in Red Rice. Figure 6This indicates that PMO not only plays a role in promoting greening during the seedling stage, but also maintains the green state and expansion capacity of functional leaves in rice during the reproductive growth stage.
[0074] PMO promotes the accumulation of photosynthetic pigments in flag leaves. After PMO treatment, the total chlorophyll in red rice flag leaves increased by 58.02%, and chlorophyll a, b, and carotenoids were all significantly increased. Figure 7 ); 9311 sword leaves showed increased total chlorophyll, chlorophyll a, and carotenoids, but decreased chlorophyll b ( Figure 7 This indicates that PMO generally promotes the formation of photosynthetic pigments in the flag leaves of different varieties, especially chlorophyll a and carotenoids, but the chlorophyll b response differs. Furthermore, PMO treatment increased the SPAD value and effective photosynthetic area of the flag leaves in both varieties. Figure 8 The results show that PMO enhances the green area of functional leaves, laying the foundation for the accumulation of photosynthetic products and grain filling in the later stages.
[0075] PMO reduced the levels of hydrogen peroxide and superoxide anions in the flag leaves. Specifically, after PMO treatment, the superoxide anion levels in the flag leaves of 9311 and Hongmi rice varieties decreased significantly by 46.10% and 14.19%, respectively (Table 1). This indicates that PMO has the ability to scavenge reactive oxygen species in tissues, effectively reducing the accumulation of superoxide anions in the flag leaves of different rice varieties, maintaining endogenous redox homeostasis, thereby delaying leaf senescence and preserving the green phenotype and photosynthetic function.
[0076] PMO analysis of the expression regulation of genes related to photosynthesis, chlorophyll synthesis, and sugar metabolism in red rice flag leaves yielded the following results: Figure 9 As shown in the figure, after PMO treatment, genes related to light harvesting and photosynthesis, such as CAB1R, CAB2R, and LHCB, as well as genes related to chlorophyll synthesis and the photosystem, such as CAO, PSAH, and OEE3, showed significant responses in rice flag leaves, indicating that PMO can promote the maintenance of photosynthetic structure and chlorophyll synthesis-related processes in flag leaves. Figure 9 Meanwhile, differential expression was also observed in sugar transport-related genes OsSUT1, OsSWEET4, and OsMST3, sucrose metabolism-related genes SPS1 and OsNIN1, and T6P / trehalose signaling and sugar / energy signaling-related genes OsTPP7, OsTPP2, SnRK1A, and HXK7. Figure 9 The above results indicate that PMO can not only increase the photosynthetic pigment content and effective leaf area of flag leaves, but may also maintain the green state and metabolic activity of functional leaves by coordinating photosynthetic carbon assimilation, sugar transport and sugar signaling regulation, thus providing a basis for the accumulation of photosynthetic products in the later stage.
[0077] In summary, PMO demonstrates good versatility in regulating the green state and expansion capacity of flag leaves: on the one hand, it promotes the accumulation of chlorophyll and carotenoids, increasing SPAD value and leaf area; on the other hand, it reduces the accumulation of reactive oxygen species such as superoxide anions, mitigating oxidative damage to functional leaves in the later stages.
[0078] The effects of spraying nano-enzyme on the H2O2 content and superoxide ion content of sword leaves are shown in Table 1. The results indicate that:
[0079] Table 1. Effects of spraying nano-enzyme on H2O2 content and superoxide ion content in sword leaves.
[0080]
[0081] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. The application of nano-enzyme mimicry PMO in regulating leaf color traits in rice, characterized in that, The nano-enzyme PMO is an SOD-type polyacrylic acid-manganese oxide nano-enzyme.
2. The application according to claim 1, characterized in that, The preparation method of the SOD-type polyacrylic acid-manganese oxide nano-enzyme is as follows: manganese sulfate aqueous solution and polyacrylic acid aqueous solution are mixed evenly and then added dropwise to ammonium hydroxide solution and stirred. The stirred solution is reacted in an oven, centrifuged to obtain the supernatant, and then dialyzed. The dialyzed solution is dried to obtain SOD-type polyacrylic acid-manganese oxide nano-enzyme.
3. The application according to claim 2, characterized in that, The concentration of the nano-enzyme PMO is 200 mg / L.
4. The application of nano-enzyme-like PMO in promoting plant growth, characterized in that, The nano-enzyme PMO is an SOD-type polyacrylic acid-manganese oxide nano-enzyme.
5. The application according to claim 4, characterized in that, The promotion of plant growth refers to increasing plant height and leaf area.
6. The application according to claim 4, characterized in that, The promotion of plant growth is achieved by increasing chlorophyll content.
7. The application of nano-enzyme mimicry PMO in regulating sugar distribution and reactive oxygen species balance in rice zebra leaves, characterized in that... The nano-enzyme PMO is an SOD-type polyacrylic acid-manganese oxide nano-enzyme.
8. The application of nano-enzyme PMO in regulating sucrose metabolism and transport-related genes and trehalose metabolism genes, characterized in that... The nano-enzyme PMO is an SOD-type polyacrylic acid-manganese oxide nano-enzyme.
9. The application according to claim 8, characterized in that, The sucrose metabolism and transport-related genes are: SPS, SPP, and SUT2; the trehalose metabolism genes are TPS2 and TPP1.
10. The application of nano-enzyme-like PMO in improving the SPAD value of rice leaves, characterized in that, The nano-enzyme PMO is an SOD-type polyacrylic acid-manganese oxide nano-enzyme.