LU503754B1 - An application of putrescine in alleviating plant growth inhibition under low nitrogen - Google Patents

Abstract

The present invention provides an application of putrescine in alleviating plant growth inhibition under low nitrogen stress. Putrescine treatment alleviated the inhibition of plant growth by low nitrogen, which was manifested in the significant increase of plant stem thickness, root length and leaf number. Meanwhile, exogenous putrescine treatment significantly increased the chlorophyll content and maximum photochemical efficiency Fv/Fm value of plants, and increased the net photosynthetic rate and nitrogen content. This showed that the appropriate concentration of putrescine could significantly alleviate the inhibitory effect of low nitrogen stress on apple plant growth, which laid a foundation for in-depth study of its functional mechanism in response to low nitrogen in apple. At the same time, the achievements may be applied in the future to guide production practices and improve the rhizosphere environment of fruit trees.

Description

An application of putrescine in alleviating plant growth inhibition under”°%7%4 low nitrogen

Technical field

The present invention relates to the technical field of pomiculture, specifically relates to an application of putrescine in alleviating plant growth inhibition under low nitrogen stress.

Background technology

Nitrogen is one of the indispensable nutrient elements for plant growth and development, and it is also the main limiting factor for plant growth. The appropriate amount of nitrogen fertilizer can meet the needs of the plant growth, improve the nutritional status of plants and promote plant growth. But, the effective utilization rate of nitrogen fertilizer by plants accounts for only 30%, and excessive application of nitrogen will enter into the surrounding soil and water, which will not only cause a series of environmental problems, but also increase the cost of agricultural production, and reduce the absorption and utilization of nitrogen by plants, indirectly causing nitrogen deficiency. Therefore, improving the nitrogen use efficiency of plants can have a profound impact on the sustainable development of agriculture.

The northwest region is the dominant production area of fruit trees in China, but the soil in orchard is high salinity and low in nutrient, resulting in poor nutrient absorption of trees.

Nitrogen is an important mineral element for fruit trees. Generally, nitrogen fertilizers are usually applied to trees to meet the needs of plant growth and development, but the amount of nitrogen fertilizer application in orchards of China are far more than that of developed countries.

For example, the amount of nitrogen fertilizer application in apple orchards in Shaanxi Province even reaches 3 to 6 times the application amount in developed countries. The excess application of nitrogen fertilizer failed to bring corresponding economic benefits and improved output; instead, it led to a series of soil and water pollution, and reduction in nitrogen absorption and utilization of fruit trees, thus to cause nitrogen deficiency. In addition, low nitrogen stress will also affect the normal physiological and metabolic activities of fruit trees, it limits the growth of root system and the above ground parts of fruit trees, such as plant height and stem thickness, and then reduces the accumulation of biomass. Therefore, how to improve the adaptability of fruit trees to the existing nitrogen supplied environment and increase the utilization rate of nitrogen fertilizer, is very important for the sustainable development of the fruit industry.

Polyamines are common small molecule plant growth regulators, mainly including putrescine (Put), spermidine (Spd), spermine (Spm), and cadaverine (Cad). They are involved in plant growth and development, sex differentiation, fruit ripening, adaptation to stresses, and other important physiological processes. For example, polyamines affect the organogenesis and the in vitro morphogenesis of the somatic embryogenesis of Lycium barbarum L.. THH 503754 accumulation of Put increased with the stress strength when subjected Robinia pseudoacacia to acid stresses with different concentration.

But the effects of exogenous polyamines on plant growth under low nitrogen stress have not been reported. By analyzing the effect of exogenous polyamines on the growth of plants under low nitrogen stress, the invention proposes an appropriate putrescine application method, which lays a foundation for the practical application of polyamines in fruit tree cultivation, and results may be applied to guide fruit production and improve the vegetative growth of fruit trees in the future.

Summary of the invention

To solve the above problems, the invention provides an application of putrescine in alleviating plant growth inhibition under low nitrogen stress.

In order to achieve the above purpose, the technical solutions of the invention is as follows:

An application of putrescine in alleviating plant growth inhibition under low nitrogen stress.

Including, the putrescine, also known as butanediamine, the molecular formula 1s

C4H12N2, the molecular weight is 88.15. The generally chemically pure (795%) is used to prepare this product.

Preferably, the concentration of putrescine in the cultivation system is 2.5~3umol/L.

Further preferably, when the concentration of putrescine is 3umol/L in the cultivation system, it significantly improves the growth of underground parts of plants under low nitrogen stress.

More preferably, the underground parts are the root architecture and root length.

Further preferably, when the concentration of putrescine is 3umol/L in the cultivation system, it significantly improves the growth of overground parts of plants under low nitrogen stress.

More preferably, the overground parts are the stem thickness and plant height.

More preferably, the purescine is used to increase the nitrogen, phosphorus, and potassium content of plants, and improve photosynthetic capacity.

More preferably, the specific application method is to add putrescine stock solution in a nitrogen stress cultivation system containing 0.1-1 mmol/L nitrogen, to make the concentration of putrescine reach 2.5-3 pmol/L.

In the application method, the putrescine stock solution is prepared by dissolving putrescine in distilled water. After preparing of the stock solution, it should be sealed and stored at room temperature in the dark. When in use, an appropriate amount of the stock solution was added to the cultivation system and mixed evenly, so that the final concentration of putrescine 9503754 3umol/L. During the cultivation, the cultivation system is changed every five days. Preferably, the cultivation system is a hydroponic system, and of course, you can also choose a soil cultivation system. The nitrogen content in the hydroponic system is 1%~10% of the nitrogen content in 1/2 Hoagland nutrient solution. The dissolved oxygen concentration in the hydroponic system is 8.0~8.5 mg/L.

Preferably, the plant is rootstock species used in Malus Mill.

Further preferably, the rootstock is Malus hupehensis.

Beneficial effects of the invention:

The invention uses the common rootstock Malus hupehensis plants as a material, through analyzing the alleviation effect of exogenous putrescine on the growth inhibition of Malus hupehensis under low nitrogen, it shows that the exogenous putrescine can effectively alleviate the growth inhibition of Malus hupehensis by low nitrogen treatment, which is evidenced by the significantly increase of plant height, stem thickness, root length, and other growth indicators, the photosynthetic capacity and the nitrogen content is also improved in plants after supplied with putrescine.

The invention treated Malus hupehensis seedlings with 3 umol/L exogenous putrescine in the hydroponic system of low nitrogen, and found that putrescine treatment alleviated the inhibition of plant growth under low nitrogen, which was manifested in the significant increase of plant stem thickness, root length, and leaf number. Meanwhile, exogenous putrescine treatment significantly increased the chlorophyll content and maximum photochemical efficiency

Fv/Fm value of plants, and increased the net photosynthetic rate and nitrogen content. This showed that the appropriate concentration of putrescine could significantly alleviate the inhibition of low nitrogen stress on apple plant growth, which laid a foundation for in-depth study of its functional mechanism in response to low nitrogen in apple. At the same time, the achievements may be applied in the future to guide fruit production and improve the rhizosphere environment of fruit trees.

The dosage of putrescine is only 3umol/L, it is very low, which can save the cost of fertilizer and alleviate the environmental pollution caused by excessive fertilizer.

Description of attached drawings

FIG 1 is the growth phenotype of Malus hupehensis plants after 35 days of treatment in

Example 2 of the invention.

FIG 2 is the detection of various indicators of the photosynthetic system of Malus hupehensis plants after 35 days of treatment in Example 2 of the invention, including SPAD value (FIG 2a), net photosynthetic rate (FIG 2b), transpiration rate (FIG 2c), stomatal conductance (FIG 2d), intercellular carbon dioxide concentration (FIG 2e), and maximuh508754 photochemical efficiency Fv/Fm (FIG 2f).

FIG 3 shows the contents of nitrogen (FIG. 3a), phosphorus (FIG. 3b), and potassium (FIG. 3c) in the roots and leaves of the Malus hupehensis plant after 35 days of treatment in Example 2 of the invention.

Specific embodiments

In order to make the purpose, technical solutions and advantages of the invention more clearly, the invention is further elaborated with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present invention but not to be limited in the present invention.

Based on the examples in the present invention, all other examples obtained by a general technician in the field without making creative results, are belonged to protection of the invention.

The experimental methods described in the following examples are conventional methods if not otherwise specified, Both the reagents and materials are commercially available on the market unless otherwise specified.

First, preparation of the reagents (1) 1/2 Hoagland nutrient solution, is composed of 0.410 g/L. Ca(N03)2-4H20, 0.253 g/L

KNO3, 0.247 g/L MgS0O4-7H20, 0.068 g/L KH2PO4-2H20, 18.350 mg/L Fe-Na-EDTA, 1.430 mg/L H3BO3, 0.110 mg/L ZnSO4-7H20, 0.905 mg/L. MnCl12-4H20, 0.040 mg/L. CuS04-SH20, and 0.045 mg/L H2MoO4-H20. The nitrogen content in the 1/2 Hoagland nutrient solution is about 6 mmol/L after conversion. (2) 1 M putrescine stock solution, which is prepared by dissolving 0.88g putrescine in 10 mL of water. After preparation of the putrescine stock solution is completed, it should be sealed and stored it at room temperature in the dark. (3) Culture medium A, which is prepared by adjusting the nitrogen content in 1/2 Hoagland nutrient solution to 1% of the original nitrogen content. (4) Culture medium B, which is prepared by adjusting the nitrogen content in 1/2 Hoagland nutrient solution to 10% of the original nitrogen content. (5) Solution A, which is prepared by adding 15 pL of 1M putrescine stock solution to 5 L of 1/2 Hoagland nutrient solution. (6) Solution B, which is prepared by adding 15 uL of 1M putrescine stock solution to 5 L of culture medium A. (7) Solution C, which is prepared by adding 15 uL of 1M putrescine stock solution to 5 L of culture medium B.

Second, the phenotypic and growth differences of Malus hupehensis response to low nitrogen/ 903754 after putrescine application.

Using the common rootstock of the Malus mill plants, Malus hupehensis, as the material, and then analyze the alleviation effect of exogenous putrescine on the growth inhibition of Malus 5 hupehensis under low nitrogen, and detect the phenotypic and growth differences of Malus hupehensis responding to low nitrogen after putrescine application. 2.1 Materials and Methods

The experiment began in November 2019, and was conducted at the test site and the stress biology of fruit trees laboratory of Northwest Agriculture & Forestry University, Yang Ling

District, Shaanxi Province.

First, the seeds of Malus hupehensis were stored at low temperature for three months, the surface of the seeds were disinfected before treatment, and then the seeds were planted into the pots. When the seedlings had 3 ture leaves, Hoagland nutrient solution was sprayed 1-2 times a week, during which the seedlings were well-watered to maintain the growth. When 8-10 ture leaves unfurled, seedlings of uniform size were selected for hydroponic treatment.

Each hydroponic pot contained 5 L of 1/2 Hoagland nutrient solution, pH was adjusted to about 6.0 using phosphoric acid. The hydroponics system was continuously supplied with oxygen, using an air pump, to maintain the dissolved oxygen concentration of the solution at 8.0-8.5mg: L-1, the solution was refreshed every 3 days. The experimental seedlings were fixed on the foam board with sponge wrapped around branches, and then the foam board was placed on the hydroponic pot. The cultivation conditions were as follows: the day temperature was 23~25 °C, the night temperature was 15~18 °C, the light source was fluorescent lamp, the photoperiod was 14 h day/10 h night, and the light intensity was 160umol-m-2-s-1.

All the Malus hupehensis seedlings were pre-cultured for 7 days, and then the seedlings were treated according to the experimental scheme in Table 1, with 60 plants per treatment group (3 replicates with 20 plants in each), and 35 days later, the growth status of seedlings under different treatments was observed and photographed. The dry and fresh weights of plants was measured by a ten-thousandth balance, the plant height was measured with a straightedge, the stem thickness was measured with a vernier caliper, the number of leaves was counted, and finally the root architecture was scanned by a root scanner (Expression 10000XL), and the parameters were analyzed by WinRHIZO/WinFOLIA (Regent Instruments Inc, Ville de Québec,

Canada) software.

Table 1 Hydroponic treatment solution

2.2 The results

After 35 days of treatment according to the protocol of treatment group I to VI, which means, after 35 days of low nitrogen stress using the hydroponics system, exogenous putrescine alleviated the growth inhibition of low nitrogen on Malus hupehensis plants (see FIG1). The effect of treatment group V was similar to the treatment group III; The effect of treatment group

VI was similar to the treatment group IV. Therefore, the effect of putrescine was compared among the treatment group I to IV.

As shown in Fig 1, the root length of plants in treatment group IV (LN+3uM Put) were significantly longer with application of putrescine. Compared with the treatment group I (CK), the stem thickness and plant height of plants in group IV (LN+3uM Put) were reduced, but they all significantly increased compared with plants in the treatment group III (LN).

In addition, the low nitrogen treatment (LN) led to a significant reduction in plant biomass accumulation, while plants under the exogenous putrescine treatment showed a slight reduction in biomass. The fresh weight was almost the same compared with the control (CK), and the dry weight merely decreased (Table 2).

Table 2 The effects of 3uM exogenous putrescine on root length, stem thickness, plant height, fresh weight, and dry weight of Malus hupehensis under low nitrogen stress

Root length 11.88+0.39c 11.99+0.29c 12.64+0.23b 15.18+0.19a

Stem thickness(mm) 2.02+0.03b 2.40+0.02a 1.81+0.02c 2.00+0.01b

Plant height(cm) 11.65+0.29a 11.85+0.19a 9.89+0.18b 10.248+0.34b

Fresh weight (g) 1.70+0.05a 1.67+0.03a 1.63+0.01a 1.65+0.03a

Dry weight (g) 0.69+0.02a 0.65+0.03a 0.46+0.02b 0.60+0.01a “Note: The data show the mean + standard deviation, the different lowercase letters indicate significant difference (P<0.05), and same below. LU503754

After low nitrogen treatment (LN), the root system of Malus hupehensis plant was stronger.

Compared with the control (CK), the total root length, surface area, volume, root tips, and lateral roots significantly increased by 40.17%, 86.25%, 103.26%, 42.47%, and 166.44%, respectively.

The treatment of exogenous putrescine (LN+3uM Put) further increased the gap between the two groups, which respectively increased by 70.71%, 115.16%, 230.50%, 60.06%, and 283.50% compared with the control (CK).

Among the four treatments, all the root parameters measured with plants in the treatment group IV (LN+3uM Put) were the highest, which were significantly higher than those of low nitrogen treatment (LN) or control treatment (CK) (Table 3).

Table 3 The effects of 3 uM exogenous putrescine on the root architecture of Malus hupehensis under low nitrogen stress

CK CK+3uM Put LN LN+3uM Put

Root length 23.9+0.33d 27.3+0.41c 33.5+0.37b 40.80. 41a

Surface area (cm?) 46.05+0.27d 55.88+0.52c 85.77+0.34b 99.08+0.49a

Volume (cm?) 0.71+0.21d 0.91+£0.22¢ 1.43+0.10b 2.33£0.19a

Number of root tips 631+5.1d 783+2.1c 899+6.1b 1010+4.9a

Number of lateral roots 879+49d 1989+69c 2342+72b 3371+89a 2.3 Conclusion

In the example of the invention, exogenous putresine can significantly alleviate the growth inhibition of Malus hupehensis plants under low nitrogen conditions, and even promote the growth of Malus hupehensis plants to restore to the status under control culture conditions.

Third, Comparison of the photosynthetic capacity of Malus hupehensis plants after supplied with putrescine under low nitrogen.

Using the common rootstock of the Malus Mill plants, Malus hupehensis, as the material, and then analyze the alleviation effect of exogenous putrescine on the growth inhibition of Malus hupehensis under low nitrogen, and detect photosynthetic capacity of Malus hupehensis responding to low nitrogen after putrescine application. 3.1 Materials and Methods

The Malus hupehensis plants were treated according to the method described in 2.1, and the average chlorophyll content (SPAD value) of the plants were determined by a portable

SPAD-502 chlorophyll meter (Konica Corporation, Japan) before and after treatment. And the maximum photochemical efficiency Fv/Fm of the leaf was determined by a Dual-PAM-100 system (Heinz Walz, Effeltrich, Germany). The CIRAS-3 portable photosynthesis systeh:}503754 (CIRAS, Amesbury, MA, USA) was used to measure the photosynthetic parameters every 5 days during the treatment, including net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, and transpiration rate. 3.2 The results

After low nitrogen treatment (LN), the chlorophyll content of Malus hupehensis leaves significantly decreased, and the chlorophyll content of plant leaves significantly increased after putrescine treatment (LN+3uM Put) (FIG 2a).

The values of the net photosynthetic rate of plants under low nitrogen continuously decreased, and was significantly lower than that of the other three treatments. After sipplied with exogenous putrescine, the net photosynthetic rate of plants remained at a high level (FIG 2b).

The same trend was reflected in the determination of stomatal conductance and transpiration rate measurement (FIG 2c-d).

The intercellular CO2 concentration was significantly higher than those of the other three treatments, while the intercellular CO2 concentration of the other three treatments was almost not changed during the 35-day treatment period (FIG 2e).

In addition, after low nitrogen treatment, the maximum photochemical efficiency Fv/Fm decreased by 12.66%. However, after exogenous putrescine treatment, the Fv/Fm value decreased by only 2.25% compared with the control, which means almost the same (FIG 2f). 3.3 Conclusion

In an example of the invention, exogenous putrescine can effectively maintain the photosynthetic system of Malus hupehensis plants under low nitrogen stress, thus to make plants have higher chlorophyll content and possess higher photosynthetic efficiency.

Fourth, the content of nitrogen, phosphorus, and potassium in Malus hupehensis after the application of putrescine under low nitrogen.

Using the common rootstock of the Malus Mill plants, Malus hupehensis, as the material, and then analyze the alleviation effect of exogenous putrescine on the growth inhibition of Malus hupehensis under low nitrogen. And measure the nitrogen, phosphorus, and potassium content of

Malus hupehensis responding to low nitrogen after putrescine application. 4.1 Materials and Methods

According to the method described in 2.1, the Malus hupehensis plants were treated with low nitrogen stress, and after the treatment, the root and leaf samples of 10 seedlings were dried and grinded thoroughly, and sieved for nitrogen, phosphorus, and potassium contents determination. We weighed 0.2 g sample into a 100 ml rigid deboiling tube, wet the sample with appropriate amount of deionized water, and then add 5 ml of concentrated sulfuric acid H2SO4,

AR, 98%) slowly, shake gently, and left it overnight. A small funnel with curved neck is placéd/°08754 on the deboiling tube, heated it slowly on the deboiling furnace. After the concentrated H2SO4 decomposed to produce white smoke, the temperature was continued to rise. When the liquid appeared brown and black, removed it from the deboiling furnace. After cooling, added 10 drops of H202 (GR, >30%) and mixed well. Continued to boil for another 20 minutes and added

H202 after cooling, and this process was repeated 2-3 times until the liquid turned to be colorless and clear. And then the samples volume was fixed to 100 ml with deionized water, and the total nitrogen and phosphorus contents were determined using a continuous flow analyzer (Auto Analyzer 3; SEAL Analytical, Norderstedt, Germany). The total potassium content was determined using a flame photometer (M410; Sherwood Scientific Ltd., Cambridge, UK). 4.2 The results

The results showed that the contents of nitrogen, phosphorus, and potassium in the leaves and roots of Malus hupehensis plants under low nitrogen treatment were significantly lower.

Compared with plants under control, the contents of nitrogen, phosphorus, and potassium in leaves and roots of plants under low nitrogen were respectively reduced by 25.15%, 49.58%, and 28.63%, and the contents of nitrogen, phosphorus, and potassium in leaves and roots of plants supplied with putrescine under low nitrogen were respectively reduced by 5.16%, 11.85%, and 5.57% (FIG 3A-C). Compared with low nitrogen, exogenous putrescine treatment significantly increased the contents of nitrogen, phosphorus and potassium in plants. 4.3 Conclusion

Low nitrogen stress reduced the contents of nitrogen, phosphorus, and potassium in Malus hupehensis plants, while exogenous putrescine treatment effectively increased the contents of nitrogen, phosphorus, and potassium in leaves and roots of plants under low nitrogen.

The above is only a good example of the present invention and is not intended to limit the present invention. Any modification, substitution, improvement, etc. made within the spirit and principles of the invention patent shall be included in the scope of protection of the invention.

Claims (10)

CLAIMS LU503754

1. An application of putrescine in alleviating plant growth inhibition under low nitrogen stress.

2. According to an application described in claim 1, its characteristics lie in that the concentration of putrescine in the cultivation system of plant growth is 2.5-3pmol/L.

3. According to an application described in claim 2, its characteristics lie in that when the concentration of putrescine is 3jumol/L in the cultivation system, it significantly improves the growth of underground parts of plants under low nitrogen stress.

4. According to an application described in claim 3, its characteristic lie in that the underground parts are the root architecture and root length.

5. According to an application described in claim 2, its characteristics lie in that when the concentration of putrescine is 3jumol/L in the cultivation system, it significantly improves the growth of overground parts of plants under low nitrogen stress.

6. According to application described in claim 5, its characteristics lie in that the overground parts are the stem thickness and plant height.

7. According to an application described in claim 3 or 5, its characteristics lie in that the purescine is used to increase the nitrogen, phosphorus, and potassium contents of plants, and improve photosynthetic capacity.

8. According to an application described in claim 2, its characteristics lie in that the specific application method is to add putrescine stock solution in a nitrogen stress cultivation system containing 0.1-1 mmol/L nitrogen, so that the concentration of putrescine reaches 2.5-3 pmol/L.

9. According to an application described in claim 1, its characteristics lie in that the plant is rootstock species used in Malus Mill.

10. According to an application described in claim 9, its characteristics lie in that the rootstock is Malus hupehensis.