CN104886053A

CN104886053A - Application of ascorbic acid to improvement of plant photosynthesis efficiency

Info

Publication number: CN104886053A
Application number: CN201510244639.9A
Authority: CN
Inventors: 李昆志; 周小华; 陈东杰; 谷照虎; 陈丽梅
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2015-05-14
Filing date: 2015-05-14
Publication date: 2015-09-09
Anticipated expiration: 2035-05-14
Also published as: CN104886053B

Abstract

The present invention discloses novel application of ascorbic acid to improvement of plant photosynthesis efficiency. The invention is as below: using ascorbic acid solution with concentration gradient of 0.1,0.5,2,5,10 mmol / L to treat different varieties of plants for 24 hours, and respectively measuring physiological and biochemical parameters of leaves; stressing by using AlCl3 with different aluminum concentration gradients and different aluminum concentration gradients containing 2 mmol / L-ascorbic acid for 24 h, and measuring the photosynthetic characteristic parameters and physiological and biochemical indices of leaves respectively; and comparing the experimental results with the control plants. After ascorbic acid administration, H2O2 accumulation in aluminum sensitive soybean and Al tolerant soybean leaf cells decrease, and H + - pump activity and stomatal aperture and photosynthetic parameters are restored and improved; and these changes help promote plant stomatal opening and enhance photosynthesis and respiration of soybean, thereby enhancing the resistance to aluminum stress of plants.

Description

Ascorbic acid is improving the application in plant photosynthesis efficiency

Technical field

The invention belongs to photosynthesis of plant field, relate to the novelty teabag of ascorbic acid, be specially ascorbic acid and improving the application in plant photosynthesis efficiency.

Background technology

Photosynthesis is plant utilization luminous energy, carbonic acid gas and water is converted into the process of organic carbon hydrate, storage power.As everyone knows, the essence of crop production is exactly that crop utilization luminous energy is the CO in air ₂be converted into organic matter storage process, dry-matter accumulation depends on the assimilation of carbon.It is generally acknowledged, the material of in crop biology output 90% ~ 95%, from photosynthetic product, only has the material of 5 ~ 10% from the mineral nutrition material of root absorption.In photosynthetic parameters, stomatal conductance is that plant and air carry out measuring of moisture and the smooth and easy degree of gas exchanges, pore is crop leaf and extraneous important channel of carrying out gas exchanges, gas and steam diffuse into Ye Nei or rising leafing all has to pass through pore outward, intercellular CO ₂the photosynthetic direct material supply of concentration impact.Therefore, each photosynthesis characteristics parameter influence photosynthesis, crop photosynthesis determines the most important factor of crop yield, and most scholar also thinks that Photosynthesis in Soybean speed etc. is proportionate with output.

Soybean (Glycine max) belongs to a cultivar in Papillionoideae (Faboideae) Glycine, therophytes, the economic crops that a kind of cultivated area is only second to paddy rice, all there is extensive plantation in China south and north, become one of most important grain and oil raw material of Chinese.In recent years, due to the use of industrialized development and a large amount of chemical fertilizer, cause acid rain frequently to occur, make the acidizing degree of acid ground and area in continuous aggravation and expand.Obstruction factor containing various plants growth in acid ground, as: low-phosphorous, acid evil, aluminium are malicious, and wherein aluminium toxicity has become a main limiting factor of crop yield in acid ground.According to statistics, the arable land in the whole world nearly 50% is in acid.And the acid ground of China is mainly throughout 15 provinces and regions, south, account for 21% of national total cultivated area.Southern area soil pH is about 4 ~ 5.5.Now in soil, slightly solubility aluminium constantly discharges with the aluminium ion form of three valence states, causes aluminium poison phenomenon.Acid-Al stress mainly causes toxic action to soybean root system, affects the photosynthetic efficiency of soybean, has certain influence to the regulation and control of Stoma of Leaves, increases stomatal resistance, causes gas exchanges inside and outside blade to be obstructed, reduce the transpiration rate of crop leaf, stomatal conductance, intercellular CO ₂concentration and Net Photosynthetic Rate, thus affect the absorption of soybean to moisture and nutrient, finally affect the growth of plant and the output of crop.Therefore, improvement and raising soybean Aluminum toxicity energy, increasing the ability that crop adapts to acid ground, is the cost-effective method solving Acid-Al stress problem.Therefore, the agent of a kind of efficient, easy, cheap and easy received raising plant photosynthesis efficiency-adjusted is found to have great reality and application value.

Summary of the invention

The object of the invention is to provide a kind of raising plant Net Photosynthetic Rate, transpiration rate, stomatal conductance, intercellular CO ₂the conditioning agent of concentration etc., namely ascorbic acid AsA is improving the application in plant photosynthesis efficiency.

In order to realize above-mentioned purpose of the present invention, technical scheme of the present invention is as follows:

1, the full seed of the responsive black soybean (black soybean for aluminum-sensitive type (Glycine max Tamba)) (calling SB in the following text) of the black soybean of resistance to aluminium profiles (black soybean for aluminum-resistant type (Glycine max Tamba)) (calling RB in the following text) in Yunnan, aluminium is selected to carry out seed disinfection, Germination under 25 DEG C of dark, treats that root grows 2cm and proceeds to 0.5mmol/L CaCl ₂balance one day in solution, transfer to Hoagland ' the s culture fluid one week of 1/4 nutrition, after growing two leaves, be transferred to Hoagland ' the s culture fluid cultivation of full nutrition;

2, when SB, RB seedling grows to four pairs of blades, the healthy and strong plant that growing way is consistent is selected, 0.1,0.5,2,5,10 mmol/L AsA solution are added to SB, RB seedling process 24 hours respectively at Hoagland ' s culture fluid, each process arranges three repetitions, get the mensuration that second pair of several from top to bottom leaf of SB, RB seedling carries out physiological and biochemical index, therefrom filter out best AsA concentration for the treatment of.

3, respectively with SB, RB seedling that the AsA process different aluminum concentration of optimization process concentration association compels, process according to the above-mentioned processing time, and get the mensuration of second pair of leaf for photosynthesis characteristics parameter and physiological and biochemical index.

Ascorbic acid AsA provided by the invention is as the stomatal conductivity agent of plant photosynthesis characterisitic parameter, easy to use, and cost is very low; This conditioning agent significantly improves the ability of plant photosynthesis characterisitic parameter, open the new way improving plant photosynthesis characterisitic parameter with conditioning agent, contribute to the molecular mechanism research that scientific worker's Ascorbic Acid AsA improves plant photosynthesis characterisitic parameter ability, soybean and the breeding of cultivating the growth of adaptation varying environment provide new Practical Research direction, also open the Growth and yield improving economical crops and provide new way.

Beneficial effect of the present invention: the conditioning agent of raising plant photosynthesis characterisitic parameter ability of the present invention, has and drops into the feature low, simple to operate, efficiency is high.Under normal temperature, AsA is the stomatal conductivity agent of more satisfactory plant photosynthesis characterisitic parameter, and AsA process can coerce H in lower minimizing soybean leaves cell in different aluminum concentration ₂o ₂accumulation, improves the H of cytoplasma membrane ⁺-pump is active, improves Net Photosynthetic Rate, transpiration rate, stomatal conductance, intercellular CO ₂concentration and stomatal aperture.AsA can recover the photosynthesis of the soybean under Acid-Al stress to a certain extent, thus improves the growth of soybean, is a kind of economic, effective crop photosynthesis promoter, significant to the economic benefit improving soybean.

Accompanying drawing explanation

Fig. 1 is the H of variable concentrations AsA treatment S B in the present invention, RB seedling 24h ₂o ₂the change of content (A figure), Stoma of Leaves aperture (B figure);

Fig. 2 is the plasma membrane H of variable concentrations AsA treatment S B in the present invention, RB seedling 24h ⁺the change of-pump activity;

Fig. 3 is the SB seedling (A figure) of 2 mmol/L AsA process 24h in the present invention, the H of RB seedling (B figure) ⁺-pump determination of activity result;

Fig. 4 is the AlCl of 0 mmol/L and 2 mmol/L AsA process variable concentrations (0,50,100,200,400 μm of ol/L) in the present invention ₃the SB seedling (A figure) of 24h, the Net Photosynthetic Rate result of RB seedling (B figure);

Fig. 5 is the AlCl of 0 mmol/L and 2 mmol/L AsA process variable concentrations (0,50,100,200,400 μm of ol/L) in the present invention ₃the SB seedling (A figure) of 24h, the transpiration rate result of RB seedling (B figure);

Fig. 6 is the AlCl of 0 mmol/L and 2 mmol/L AsA process variable concentrations (0,50,100,200,400 μm of ol/L) in the present invention ₃the SB seedling (A figure) of 24h, the stomatal conductance result of RB seedling (B figure);

Fig. 7 is the AlCl of 0 mmol/L and 2 mmol/L AsA process variable concentrations (0,50,100,200,400 μm of ol/L) in the present invention ₃the SB seedling (A figure) of 24h, the intercellular CO of RB seedling (B figure) ₂concentration results.

Embodiment

Below by embodiment and accompanying drawing, the present invention is described in further detail, but scope is not limited to described content.In embodiment, method if no special instructions, and operation is carried out routinely, and use reagent is the reagent that routine is purchased reagent or prepared according to a conventional method if no special instructions.

embodiment 1:the cultivation of SB, RB plant and process, concrete steps are as follows:

1, experiment material is SB, RB seedling

After selecting full SB, RB seed disinfection vernalization, Germination under 25 DEG C of dark, treats that root grows 2cm and proceeds to 0.5mmol/L CaCl ₂balance one day in solution, transfer to Hoagland ' the s culture fluid one week of 1/4, be transferred to the cultivation of Hoagland ' s culture fluid after growing two leaves, test for this when seedling grows to four pairs of leaves.

2, the AsA treatment fluid of variable concentrations (0.1,0.5,2,5,10 mmol/L) is configured, the AlCl of variable concentrations (0,50,100,200,400 μm of ol/L) ₃treatment fluid.

3, respectively with above-mentioned AsA gradient concentration treatment S B, the process of RB seedling 24 hours, H is carried out in sampling ₂o ₂content, stomatal aperture, cytoplasma membrane H ⁺the mensuration of-pump activity index content.

4, with the AlCl of 2 mmol/L AsA process variable concentrations (0,50,100,200,400 μm of ol/L) ₃sB, RB seedling, with AlCl ₃sB, RB seedling that Hoagland ' the s culture fluid that concentration and ASA concentration are 0 is cultivated is blank, processes 24 hours, and the mensuration of every physiological and biochemical index is carried out in sampling, and each process arranges three repetitions; Experimental session, Hoagland ' s culture fluid pH4.5, temperature Change is at 13 ~ 22 DEG C round the clock, and illumination and dark processing time are set as 12h/12h.

embodiment 2:sB, RB blade in embodiment 1 after the 3rd step process is adopted to carry out H ₂o ₂content, Stoma of Leaves aperture and plasma membrane H ⁺the determination of activity of-pump

1, H ₂o ₂assay: adopt xylenol orange method.Take fresh plant blade, add according to the ratio of material 1g and precooling acetone extraction agent 1mL, add after a little quartzite sand grind becomes homogenate, proceed to centrifuge tube in 12000g, 4 DEG C of centrifugal 20min, discard residue, supernatant is sample extracting solution; Use ddH respectively ₂o reagent preparation A (includes 3.3 mmol/L FeSO ₄, 3.3 mmol/L (NH ₄) ₂sO ₄, 412.5 mmol/L H ₂sO ₄) and reagent B (including 165 μm of ol/L xylenol orange, 165 mmol/L sorbierites), use front reagent A and reagent B according to 1:10(v/v) ratio mixing form working reagent; This working reagent and H ₂o ₂liquid to be measured is according to 2:1(v/v) ratio mixing, 30 DEG C of water-baths colour developing 30min, measure OD value in 560nm place, calculating H ₂o ₂content (Figure 1A).

As can be seen from Figure 1A, with the AsA process of variable concentrations after 24 hours, the H of SB, RB soybean leaves ₂o ₂content with do not compare the minimizing all had in various degree with the plant of AsA process; 2 mmol/L AsA processed group H ₂o ₂content reduces at most, and AsA concentration raises again, H ₂o ₂content no longer includes significant change.

2, Stoma of Leaves aperture measures: the blade having processed 24 hours in Example 1 the 3rd step, tears and takes off epidermis, be placed on cover glass, drip a physiological saline, covered, in times Microscopic observation measure stomatal aperture, each concentration random measurement 10, average (Figure 1B).Photosyntheticly be determined at 9: 00 ~ 10: 00 and carry out.

From Figure 1B, variable concentrations AsA treatment S B, RB soybean leaves stomatal aperture have raising in various degree, but the increase degree of 2 mmol/L AsA to soybean leaves stomatal aperture is maximum.

3, plasma membrane H ⁺-pump determination of activity step is as follows:

Use BTP to adjust pH to 6.0 containing 5 mmol/L BTP/MES (pH 6.0), 12 μm of ol/L AO, 300 mmol/L KCl, 250 mmol/L sucrose, 0.5 mmol/L EGTA(in (1) 1.5 ml reaction system), 1 mmol/L NaN ₃, 1 mmol/L Na ₂moO ₄, 50 mmol/L KNO ₃0.05% Brij-35 (w/v) and 100 μ g plasmalemma proteins, adding abstergent Brij-35 makes original position film overturn, and after reaction mixture at room temperature places 20 min, adds 5 mmol/L ATP/BTP(pH=6.0) to start reaction;

(2) with reactant liquor zeroing contrast, record per minute OD value, mensuration acridine orange is 20 minutes internal absorbance quencher speed at 492 nm place, reflects that the soybean leaves cytoplasma membrane of different AsA concentration pumps H by quencher speed ⁺ability, i.e. H ⁺-pump activity (Fig. 2).

From Fig. 2, variable concentrations AsA treatment S B, RB soybean leaves pump H ⁺ability have raising in various degree, but 2 mmol/L AsA pump H to soybean leaves ⁺ability increase degree maximum.

As seen from Figure 1, under Acid-Al stress environment, compare with SB, RB blade not with AsA process with SB, RB blade of AsA process, H ₂o ₂accumulation obviously declines (Figure 1A), Stoma of Leaves aperture increases (Figure 1B), H ⁺-pump is active obviously raises (Fig. 2).This illustrates that the AsA of 2 mmol/L consumes H in soybean leaves cell ₂o ₂, and SB, RB blade blade stomatal aperture, plasma membrane H under making Acid-Al stress ⁺-pump activity is gone up.

embodiment 3:according to above-mentioned SB, RB blade cell H ₂o ₂content, stomatal aperture, cytoplasma membrane H ⁺-pump active quantities measurement result, filters out the optimization process concentration that 2 mmol/L are AsA.In order to verify under different aluminum concentration (0,100 μm of ol/L) is coerced, the photosynthesis characteristics parameter of 2 mmol/L AsA to different cultivars soybean has larger facilitation, the process of embodiment 1 the 3rd step is carried out with optium concentration 2 mmol/L AsA treatment fluid, be that 0 respectively treatment S B, RB seedling are for contrast with AsA concentration, process 24h measures plant leaf plasma membrane H ⁺-pump activity (Fig. 3 A, 3B).

As can be seen from Figure 3, after using 2 mmol/L AsA process, the H of soybean leaves ⁺-pump with do not use the aluminum concentration of AsA assist compel under soybean plant strain compare the raising had in various degree, plasma membrane H ⁺-pump is the important regulatory factor in the open activity of plant stomata, H ⁺-pump increased activity all contributes to the increase of stomatal aperture or degree of leading, thus promotes stomatal opening.

embodiment 4:the treatment S B of embodiment 1 the 4th step, RB soybean Net Photosynthetic Rate (Fig. 4), transpiration rate (Fig. 5), stomatal conductance (Fig. 6) and intercellular CO ₂concentration determination (Fig. 7)

Use the portable photosynthetic transpiration instrument of Yaxin-1102 of Beijing Ya Xinli instrument Science and Technology Ltd., measure to specifications.Photosyntheticly be determined at 9: 00 ~ 10: 00 and carry out, measure repetition 5 times.

From Fig. 4, Fig. 5, employ ASA process (0,50,100,200,400 μm of ol/L) the variable concentrations AlCl of 2 mmol/L ₃sB, RB blade with do not use the different aluminum concentration of AsA assist compel under soybean plant strain compare, along with the increase of aluminum concentration, Net Photosynthetic Rate, the transpiration rate of its blade are all declining, and SB rate of descent is larger than RB, and all have increase in various degree, AsA process 100 μm of ol/L AlCl by the Net Photosynthetic Rate, transpiration rate etc. of soybean after AsA process ₃the Net Photosynthetic Rate of SB be even 1.05 times of the control group of not compeling with aluminium association, but with AsA process 400 μm of ol/L AlCl ₃net Photosynthetic Rate recover the strongest, and SB increases more than RB, is be not about 300% by the Net Photosynthetic Rate of the SB of AsA process with the SB of AsA process.And the transpiration rate of 100 of AsA process μm of ol/L SB is do not assist the control group of compeling with aluminium 1.26 times, but with AsA process 400 μm of ol/L AlCl ₃transpiration rate recover at most, and SB recovers more than RB, and the SB of AsA process compares not with the transpiration rate increase about 175% of the SB of AsA process.

From Fig. 6, Fig. 7, employ AsA process (0,50,100,200,400 μm of ol/L) the variable concentrations AlCl of 2 mmol/L ₃sB, RB blade with do not use the different aluminum concentration of ASA assist compel under soybean plant strain compare, along with the increase of aluminum concentration, the stomatal conductance of its blade, intercellular CO ₂concentration is all in decline, and SB rate of descent is larger than RB, and all has increase in various degree with the stomatal conductance of ASA process, returns to 0.96 times of not urgent with aluminium association control group with the SB of AsA process 100 μm of ol/L, but with AsA process 400 μm of ol/L AlCl ₃stomatal conductance recover the strongest, and SB recovers more than RB, and the SB of AsA process compares not with the stomatal conductance increase about 187% of the SB of AsA process.And the intercellular CO of AsA process ₂concentration all has increase in various degree, and the SB of AsA process 100 μm of ol/L returns to 0.98 times of the control group of not compeling with aluminium association, but with AsA process 400 μm of ol/L AlCl ₃intercellular CO ₂concentration is recovered at most, and SB recovers more than RB, compares not with the intercellular CO of the SB of AsA process with the SB of AsA process ₂concentration increase about 41%.

Claims

1. ascorbic acid is improving the application in plant photosynthesis efficiency.