CN111567338B - A method for improving photosynthetic carbon assimilation ability of maize under high temperature stress - Google Patents
A method for improving photosynthetic carbon assimilation ability of maize under high temperature stress Download PDFInfo
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- CN111567338B CN111567338B CN202010449262.1A CN202010449262A CN111567338B CN 111567338 B CN111567338 B CN 111567338B CN 202010449262 A CN202010449262 A CN 202010449262A CN 111567338 B CN111567338 B CN 111567338B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/20—Cereals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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- Forests & Forestry (AREA)
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- Botany (AREA)
- Agronomy & Crop Science (AREA)
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- Health & Medical Sciences (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明公开了一种提高玉米高温胁迫下光合碳同化能力的方法，属于生物科学技术领域，本发明提供外源海藻糖在提高高温胁迫下玉米光合碳同化能力中的方法，并给出其浓度为0.5mmol/L；本发明表明，通过上调磷酸烯醇丙酮酸羧化酶、NADP‑苹果酸脱氢酶、NADP‑苹果酸酶、丙酮酸磷酸二激酶和Rubisco小亚基的转录水平，同时促进包括PEPC、NADP‑MDH、NADP‑ME和RUBPCase等C4途径关键酶的活性，外源海藻糖可以有效地提高光合碳同化的能力并提高玉米幼苗中的碳水化合物含量。
The invention discloses a method for improving the photosynthetic carbon assimilation ability of maize under high temperature stress, belonging to the technical field of biological sciences. The invention provides a method for improving the photosynthetic carbon assimilation ability of maize under high temperature stress with exogenous trehalose, and the concentration thereof is given. is 0.5mmol/L; the present invention shows that by up-regulating the transcription levels of phosphoenolpyruvate carboxylase, NADP-malate dehydrogenase, NADP-malate enzyme, pyruvate phosphokinase and Rubisco small subunit, and at the same time By promoting the activities of key C4 pathway enzymes including PEPC, NADP-MDH, NADP-ME and RUBPCase, exogenous trehalose can effectively enhance the ability of photosynthetic carbon assimilation and increase the carbohydrate content in maize seedlings.
Description
Technical Field
The invention relates to the technical field of bioscience, in particular to a method for improving the assimilation capacity of photosynthetic carbon under the high-temperature stress of corn.
Background
In recent years, with the increase in greenhouse effect, the occurrence of abnormally high temperature weather has become more frequent, and the global temperature is expected to rise by 1.8 to 4 ℃ by 2100 years. High temperature is an important environmental factor affecting plant growth and development. It has been reported that for every 1 ℃ increase in temperature, crop yield can be reduced by 17%. Photosynthesis is extremely sensitive to high temperatures that can destroy photosynthetic pigments, photosynthetic systems, photosynthetic electron transport, and photosynthetic carbon assimilation. The response of photosynthesis in C4 plants at room temperature is similar to that in C3 plants, but the optimum temperature for C4 plants is higher than that of C3 plants, which can be attributed to CO in C4 plants2The concentration mechanism reduces the photosynthesis inhibition of the C4 plant. Photosynthetic carbon assimilation of maize (a C4 crop) has specialized CO2The concentration mechanism, comprising four stages (carboxylation, reduction, decarboxylation and regeneration) and the calvin cycle. Phosphoenolpyruvate Carboxylase (PEPC), NADP-malate dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME) and Pyruvate Phosphate Dikinase (PPDK) are CO2The key enzyme in the concentration mechanism, called the C4 pathwayAn enzyme. The key enzyme of the calvin cycle is ribulose 1, 5-bisphosphate carboxylase (RUBPCase). However, when the leaf temperature is higher than 38 ℃, the photosynthesis of corn is also disturbed. For example, at elevated temperatures, PEPC activity and PEP regeneration are significantly reduced, whereas at temperatures above 32.5 ℃, the activation state of Rubisco is reduced and at 45 ℃ almost completely inactivated. Although their optimal balance is important for the normal function of photosynthesis, it also interferes with the balance between different enzymes under heat stress.
Therefore, there is a need to improve the ability of corn to adapt to heat stress to cope with harsh environments.
Disclosure of Invention
The invention aims to provide a method for improving the photosynthetic carbon assimilation capability of corn under high-temperature stress so as to solve the problems in the prior art, and the invention discovers that exogenously provided trehalose is beneficial to improving the photosynthetic carbon assimilation capability and then increasing the content of carbohydrate.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for improving the assimilation ability of photosynthetic carbon under high-temperature stress of corn, which adds exogenous trehalose in a corn seedling stage.
Further, the concentration of the exogenous trehalose is 0.5 mMol/L.
Further, the exogenous trehalose improves carbon assimilation ability by increasing the activity of key enzymes in the photosynthetic carbon assimilation process.
Further, the key enzymes include PEPC, NADP-MDH, NADP-ME and Rubi sco.
The invention discloses the following technical effects:
the present inventors investigated the influence of exogenous trehalose on carbohydrate content, gas exchange parameters, C4 pathway enzymes and ribulose 1, 5-bisphosphate carboxylase (RUBPCase; EC 4.1.1.39) and the transcription levels of genes encoding these enzymes in maize seedlings under heat stress conditions. The results show that exogenous trehalose can effectively improve the capacity of photosynthetic carbon assimilation and the carbohydrate content in maize seedlings by up-regulating the transcription levels of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), NADP-malate dehydrogenase (PEPC; EC 4.1.1.31), NADP-MDH (EC 1.1.1.82), NADP-malic enzyme (NADP-ME; EC 1.1.1.40), pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) and Rubis co small subunit (Rubisco-SSU) while promoting the activity of key enzymes of the C4 pathway including PEPC, NADP-MDH, NADP-ME and RUBPCase.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph showing the effect of high temperature stress of different degrees on the maximum photochemical efficiency of photosystem II of maize seedling leaves of different treatment groups, wherein H38 deg.C, H40 deg.C, H42 deg.C and H45 deg.C represent high temperature treatment of maize seedlings at 38 deg.C, 40 deg.C, 42 deg.C and 45 deg.C for 1 day, respectively; c: high-temperature treatment is not carried out; HR 0: treating at high temperature for 1 day; HR 1: recovering for 1 day at normal temperature after high temperature stress; represents P < 0.05; represents P < 0.01;
FIG. 2 is a graph showing the effect of different concentrations of exogenous trehalose on the maximum photochemical efficiency of photosystem II of maize seedlings from different treatment groups, wherein CK, T0.5, T1.5, T10, T20 and T30 represent 3 days of culture with nutrient solutions containing 0mM (control), 0.5mM, 1.5mM, 10mM, 20mM and 30mM trehalose, respectively; c: high-temperature treatment is not carried out; HR 0: high temperature treatment (42 ℃) for 1 day; HR 1: recovering for 1 day at normal temperature after high temperature stress; different letters represent significant differences (P < 0.05);
FIG. 3 is a graph of the effect of exogenous trehalose on leaf gas exchange parameters of maize seedlings from different treatment groups, where Pn: a net photosynthetic rate; gs: conductivity of the air hole; ci: intercellular CO2Concentration; tr: a transpiration rate; Ci/Ca: intercellular CO2With ambient CO2The ratio of the partial pressures; WUE: water utilization efficiency; CK: a control group; t: a trehalose pretreatment group; c: high-temperature treatment is not carried out; HR 0: height ofWarming for 1 day; HR 1: recovering for 1 day at normal temperature after high temperature stress; different letters represent significant differences (P)<0.05)；
FIG. 4 is the effect of exogenous trehalose on carbon assimilation key enzyme activity of maize seedling leaves of different treatment groups, wherein PEPC: phosphoenolpyruvate carboxylase; NADP-MDH: NADP malate dehydrogenase; NADP-ME: NADP malic enzyme; PPDK: pyruvate phosphate dikinase; ru bisco: ribulose 1,5 bisphosphate carboxylase/oxygenase; CK: a control group; t: a trehalose pretreatment group; c: high-temperature treatment is not carried out; HR 0: treating at high temperature for 1 day; HR 1: recovering for 1 day at normal temperature after high temperature stress; different letters represent significant differences (P < 0.05);
FIG. 5 shows the effect of exogenous trehalose on the expression of leaf carbon assimilation key enzyme genes of maize seedlings of different treatment groups, wherein PEPC: phosphoenolpyruvate carboxylase; NADP-MDH: NADP malate dehydrogenase; NADP-ME: NADP malic enzyme; PPDK: pyruvate phosphate dikinase; Rubisco-LSU: ribulose 1,5 diphosphonate large subunit; Rubisco-SSU: ribulose 1,5 diphosphoric small subunit; CK: a control group; t: a trehalose pretreatment group; c: high-temperature treatment is not carried out; HR 0: treating at high temperature for 1 day; HR 1: recovering for 1 day at normal temperature after high temperature stress; different letters represent significant differences (P < 0.05).
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
To verify the beneficial effects of the present invention, the following experiments were performed:
1 materials and methods
1.1 cultivation of the test Material
Selecting healthy and plump corn (Zea mays L.) seed (Shenke glutinous No. 1), washing with clear water, covering with 4 layers of white gauze soaked with water for pregermination for 2-3 days, placing the corn seed with good germination condition in plastic tray with small holes, and culturing with clear water for 16h under illumination intensity of 800 umol.m-2·s-1The temperature was 25/20 deg.C (day/night). When the corn seedlings grow to 1 leaf and 1 heart, the clear water is replaced by Hoagland nutrient solution for continuous culture.
1.2 high temperature stress treatment
When the third leaf of the maize seedling grows, the maize seedling is placed into a light culture box and subjected to high-temperature stress treatment (38, 40, 42, 45 ℃ C.; humidity is 38%) for 1 day (13 h in the day and 11h in the night) to form a high-temperature treatment group. Then, the maize seedlings after high temperature stress were placed under the condition of 25/20 deg.C (day/night) and cultured for 1 day to obtain a high temperature recovery group. The extent of the high temperature stress treatment can be selected by measuring the fluorescence parameters of the third leaf of maize.
1.3 trehalose treatment
When the third leaf of the corn seedling grows, the seedling is cultured for 3 days by nutrient solution added with trehalose (0.5, 1.5, 10, 20 and 30mM) with different concentrations, and the seedling is a trehalose treatment group. Seedlings cultured with nutrient solution without trehalose addition served as control group. Then, both groups of seedlings were simultaneously placed in a light incubator at a high temperature (42 ℃ C.; humidity 38%) for 1 day (13 hours in the day and 11 hours in the night) to be a high-temperature treatment group. Then, the maize seedlings after high temperature stress were placed under the condition of 25/20 deg.C (day/night) and cultured for 1 day to obtain a high temperature recovery group. The concentration of trehalose treatment can be selected by measuring the fluorescence parameter of the third leaf of maize.
1.4 determination of chlorophyll fluorescence parameters
Dark adaptation of the sample for 20-30min, and determination of chlorophyll fluorescence parameters F of different treatment groups of leaves with pulse amplitude modulation chlorophyll fluorescence apparatus (DUAL-PAM-100, Walz, Germany)0(minimum chlorophyll fluorescence intensity under dark adaptation), Fm (maximum chlorophyll fluorescence intensity under dark adaptation), and the maximum photochemical efficiency of photosystem II was calculated: Fv/Fm ═ (Fm-Fo)/Fm.
1.5 determination of carbohydrate content
The determination of carbohydrate content was carried out according to the method of Jang et al (2003) with minor modifications. 5g of the leaf were ground into a fine powder with liquid nitrogen, and further ground into a homogenate by adding 15mL of ultrapure water, and placed in a 50mL stoppered tube in a boiling water bath for 15min, followed by centrifugation at 3000g for 15 min. The supernatant was collected and filtered through a 0.45-. mu.m filter. The carbohydrates in the leaves of maize seedlings were quantitatively analyzed by HPLC (Agilent 1260, USA) using a Shodex Asahi pak NH 2P-504E column (4.6X 250mm) using an evaporative light scattering detector, with a sample size of 10. mu.L. Trehalose, fructose, mannitol, glucose and sucrose (Sigma, st. louis, USA) were used as standards for commercial products.
1.6 determination of gas exchange parameters
Using LI-6400 photosynthesizer (LI-COR, USA) at 25 deg.C and 800 μmol m-2s-1And CO2The concentration is about 400. mu. mol-1Measured under the conditions ofAnd the gas exchange parameters of the leaves of the corn seedlings in the same treatment group.
1.7 determination of carbon assimilation Key enzyme Activity
0.5g of differently treated leaves of maize seedlings are placed in a mortar and 3mL of a precooled 25mM HEPES-KOH (pH 7.5) enzyme extract [10mM MgSO 5 ] is added41mM EDTA, 5mM DTT, 1mM PMSF, 5% (w/v) PVP and 0.05% (v/v) Triton X-100]Fully grinding by using liquid nitrogen, filtering homogenate by using 2 layers of gauze, centrifuging the homogenate by 5min at the temperature of 4 ℃ at 14000g, taking supernatant fluid which is enzyme crude extraction liquid, and storing the supernatant fluid at the temperature of 0 ℃ for later use.
The change in OD value at 340nm per minute was measured as 1 enzyme activity unit using an ultraviolet spectrophotometer (FLUOstar Omega, BMG Labtech, Germany). Enzyme activity reaction solution of PEPC: 25mM Tris-HCl buffer (pH 8.0), 1mM EDTA, 5mM MgCl2，10mM NaHCO35mM DTT, 5mM glucose-6-phosphate, 5mM PEP, 0.2mM NADH, 2U MDH. Enzyme activity reaction solution of NADP-MDH: 25mM Tricine-KOH buffer (pH 8.3), 150mM KCl, 2mM OAA, 0.2mM NADPH, 5mM DTT. Enzyme activity reaction solution of NADP-ME: 25mM Tris-HCl buffer (pH 8.3), 1mM EDTA, 5mM DTT, 2.5mM L-malic acid, 0.25mM NADP+Adding 5mM MgCl2The reaction was started. Enzyme activity reaction solution of PPDK: 25mM HEPES-KOH buffer (pH 8.0), 1mM EDTA, 10mM NaHCO3，5mM DTT，8mM MgSO42mM pyruvate, 1mM ATP, 5mM (NH)4)2SO41mM glucose 6-phosphate, 2.5mM K2HPO40.2mM NADH, 0.5U PEPC, 2U MDH. Enzyme activity reaction liquid of Rubisco: 25mM HEPES-KOH buffer (pH 7.8), 1mM EDTA, 10mM NaHCO3，5mM DTT，20mM MgCl21mM RuBP, 0.2mM NADH, 5mM ATP, 5mM creatine phosphate, 2U creatine phosphokinase, 2.8U glyceraldehyde-3-phosphate dehydrogenase, 2.0U 3 phosphoglycerate kinase. And (3) calculating enzyme activity: u ═ V)/(Δ t · Vs ·, W), where OD: absorbance at 340 nm; v: the total volume of the sample extract; Δ t: enzymatic reaction time; vs: measuring the volume of the extracted solution of the sample; w: sample mass.
1.8 q-PCR detection of carbon assimilation Key enzyme genes
Extraction of RNA: leaves of maize seedlings from different treatment groups were used for total RNA extraction using RNAioso plus (Takara, Japan) according to the instructions. The method comprises the following specific steps:
1. the corn leaves were placed in a 2mL EP tube and 4 magnetic beads and 1mL of RNAioso Plus (Takara) extract were added, and the homogenizer was shaken for 30s and then allowed to stand for 5 min.
2. The EP tube with the extract was transferred to a pre-cooled refrigerated centrifuge and centrifuged at 12000g for 5min at 4 ℃.
3. The supernatant was taken to a new EP tube and 200. mu.L chloroform was added, shaken well and left to stand for 5 min. Followed by centrifugation at 12000g for 15min at 4 ℃.
4. The supernatant was taken into a new EP tube and 200. mu.L of isopropanol solution was added and shaken gently and left to stand at-20 ℃ for 30 min.
5. At 4 ℃ 12000g for 10min, the supernatant was discarded and the pellet was retained.
6. 1mL of 75% ethanol was added, the pellet was resuspended, and centrifuged at 12000g for 2min at 4 ℃.
7. The supernatant was decanted, allowed to stand at room temperature, the precipitate was air-dried, 10. mu.L of DEPC water was added to dissolve the RNA, and the RNA was stored at-20 ℃ for further use.
Reverse transcription of RNA: after the extraction of sample RNA was completed, cDNAs were synthesized according to the instruction manual of cDNA Synthesis kit (Ye ason, Shanghai). The method comprises the following specific steps:
1. removal of genomic DNA
RNase free ddH2O to 16μL
4×gDNA wiper Mix 4μL
1pg-1000ng of template RNA Total RNA
After heat shock at 42 ℃ for 2min, the cells were placed on ice.
2. Configuration of reverse transcription System
To the above reaction solution was added 4. mu.L of 5 Xsuper Mix II.
3. Setting of reverse transcription program
Keeping the temperature at 25 ℃ for 10 min; heating at 42 deg.C for 30 min; heat shock at 85 deg.C for 5 min. The DNA product of c was obtained and stored in a freezer at-20 ℃ for further use.
q-PCR detection of expression of carbon assimilation key enzyme genes: c to be synthesizedThe DNA was diluted 10-fold and used as a template for q-PCR. The primer sequences of the target genes are shown in Table 1 and 2 by taking maize actin2 as the reference gene-ΔΔCTThe method calculates the relative expression quantity of the carbon assimilation key enzyme gene. Using a q-PCR instrument (CFX96, Bio-Rad, USA) according to HieffTM q-PCR
The q-PCR experiment was performed in the instruction manual of the Green Master Mix (No Rox Plus) kit (Yeason, Shanghai).
Reaction system (20 μ L): (BIO-RAD, USA)
The q-PCR program was set up as follows: firstly, pre-denaturation is carried out for 5min at 95 ℃; ② denaturation at 95 ℃ for 10 s; ③ annealing at 55 ℃ for 20 s; extension for 20s at 72 ℃, and carrying out 40 cycles on the- (III). The dissolution curve analysis was 65 ℃ to 95 ℃ and 5s higher per liter at 5 ℃.
TABLE 1 primers for maize carbon assimilation key enzyme genes
1.9 data analysis
All data were statistically analyzed using SPSS 21.0(SPSS, Chicago, USA) and a one-way analysis of variance (ANOVA) was performed on each individual sample according to Tukey's test with significance levels set at P.ltoreq.0.05 (significant) and P.ltoreq.0.01 (very significant). All experiments were performed in at least 3 replicates and all data are presented as mean ± standard deviation.
2 results
2.1 Effect of varying degrees of high temperature stress on Fv/Fm
The photochemical efficiency of PSII is reduced due to high temperature stress, resulting in a decrease of Fv/Fm. Therefore, the invention selects a proper high temperature stress temperature by respectively measuring the reduction degree of Fv/Fm under the high temperature stress of 38 ℃, 40 ℃, 42 ℃ and 45 ℃. The results are shown in FIG. 1: the Fv/Fm is not affected by the high-temperature stress at 38 ℃; when the temperature rises to 40 ℃, the Fv/Fm is remarkably reduced and can return to the original level in the recovery period; when the temperature rises to 42 ℃, the Fv/Fm is reduced more obviously, and the Fv/Fm cannot return to the original level in the recovery period; when the temperature was further increased to 45 ℃ almost no Fv/Fm values could be detected. The larger the degree of Fv/Fm reduction, the more severe the high temperature stress. Therefore, the following experiment will select a high temperature stress of 42 ℃ for the following study.
2.2 Effect of different concentrations of exogenous trehalose on Fv/Fm
Trehalose is an effective plant stress protectant, and this experiment will select an optimal exogenous trehalose pretreatment concentration that protects the photochemical efficiency of PSII by determining the increase of Fv/Fm of corn seedlings pretreated with 0mM (control), 0.5mM, 1.5mM, 10mM, 20mM and 30mM exogenous trehalose, respectively, as compared to the control (0mM exogenous trehalose) under high temperature stress at 42 ℃. The experimental result is shown in figure 2, and Fv/Fm can be respectively improved by 24% and 22% by exogenously adding 0.5mM and 1.5mM trehalose under high-temperature stress; exogenously added 10mM and 20mM trehalose were not different from Fv/Fm in the control group; exogenous addition of 30mM trehalose significantly reduced Fv/Fm. In addition, only 0.5mM trehalose pretreatment promoted the Fv/Fm rebound during the normal temperature recovery period following high temperature stress (FIG. 2). Thus, the following experiment will be conducted with trehalose pretreatment at 0.5 mM.
2.3 Effect of exogenous trehalose on carbohydrate content under high temperature stress
Since trehalose can serve as a central coordinator of carbohydrate synthesis and circulation in plants, this experiment explored the effect of 0.5mM trehalose pretreatment on the endogenous trehalose and other carbohydrate content of maize seedlings. The experimental results of table 2 show that the contents of trehalose, fructose, mannitol, glucose and sucrose in the leaves of maize seedlings in the 0.5mM trehalose pretreated group were increased by 32%, 68%, 12%, 94% and 88%, respectively, at the room temperature stage, compared to the control group. High temperature stress significantly reduced the carbohydrate content, but exogenous trehalose pretreatment increased the endogenous trehalose content by up to 91% compared to the control group under high temperature stress, while also significantly increasing the content of the other four carbohydrates (table 2). Likewise, during the normal temperature recovery period after high temperature stress, exogenous trehalose pretreatment also increased the carbohydrate content in the leaves of maize seedlings (table 2).
TABLE 2 Effect of exogenous trehalose on the carbohydrate content of maize seedlings leaves of different treatment groups
Trehalo: trehalose; fructose: fructose; mannitol: mannitol; glucose: glucose; sucross: sucrose; CK: a control group; TRE: a trehalose pretreatment group; h: treating at high temperature for 1 day; HR: after high temperature stress, the product was recovered at room temperature for 1 day. Different letters represent significant differences (P < 0.05).
2.4 Effect of exogenous trehalose on gas exchange parameters under high temperature stress
In order to investigate whether the increased accumulation of carbohydrates by exogenous trehalose pretreatment was associated with an increase in leaf photosynthetic rate, the present invention determined the gas exchange parameters of maize seedling leaves. In the high-temperature stress stage, Pn, Gs, Tr and WUE in the exogenously added trehalose group and the exogenously added trehalose group are all reduced remarkably, but Pn and WUE in the exogenously added trehalose group are obviously higher than those in the exogenously added trehalose group, which shows that the exogenously added trehalose can remarkably improve the net photosynthesis capacity and the water utilization efficiency of the heat-stressed corn seedlings (fig. 3A, B, D and F). Notably, Ci and Ci/Ca in the control group were significantly increased under high temperature stress compared to the control group at the normal temperature stage; the Ci and Ci/Ca in the exogenously added trehalose group under high temperature stress is not different from the exogenously added trehalose group at the normal temperature stage (FIG. 3C, E). Therefore, exogenous trehalose obviously improves the carbon assimilation capability of corn seedlings under high-temperature stress, and accelerates CO2The utilization efficiency of (2).
2.5 Effect of exogenous trehalose on carbon assimilation Key enzyme Activity under high temperature stress
The activities of PEPC, NADP-MDH, NADP-ME, PPDK and Rubisco play a key role in the carbon assimilation process of photosynthesis of leaves of maize seedlings. High temperature stress significantly inhibited their activity (fig. 4). There was no significant difference in the activities of these enzymes between the control group and the trehalose-pretreated group at the time of the normothermic period (FIG. 4). However, trehalose pretreatment significantly enhanced the activity of PEPC, NADP-MDH, NADP-ME, and Rubisco during the high temperature phase (FIG. 4A, B, C, E). Trehalose pretreatment significantly enhanced the activity of PEPC, NADP-ME and Rubisco during the normothermic recovery phase following high temperature stress (fig. 4A, C, E). In conclusion, it can be concluded that trehalose pretreatment can improve the carbon assimilation ability of corn seedlings by increasing the activity of key enzymes in the carbon assimilation process of photosynthesis, thereby improving the heat resistance thereof.
2.6 influence of exogenous trehalose on expression of carbon assimilation Key enzyme genes under high temperature stress
The influence of trehalose pretreatment on the expression of carbon assimilation key enzyme genes was further analyzed by q-PCR experiments. As can be seen from FIG. 5, in the control group, high temperature stress decreased the gene expression of PEPC, PPDK and Rubisco-SSU, increased the gene expression of NADP-ME, and had almost no effect on the gene expression of NADP-MDH and Rubisco-LSU. At the normal temperature stage, trehalose pretreatment significantly increased the gene expression of PPDK and Rubisco-LSU (fig. 5D, E). During the high temperature stress stage, trehalose pretreatment significantly increased the gene expression of PEPC, NADP-MDH, NADP-ME, PPDK and Rubisco-SSU (FIG. 5A, B, C, D, F). At the normothermic recovery stage following high temperature stress, trehalose pretreatment only increased the gene expression of Rubisco-SSU (fig. 5F).
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
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