A method for evaluating nitrate supply and demand relationships and partitioning of nitrate assimilation products in woody plants

Abstract

The application discloses a method for evaluating nitrate supply-demand relationship and nitrate assimilation product distribution of woody plants, wherein the woody plant seedlings are planted in a nutrient solution containing only nitrate nitrogen, the dry weight, nitrogen content and stable nitrogen isotope value of the leaves, stems and roots of the woody plant seedlings before and after the experiment are measured, the stable nitrogen isotope value of the whole plant scale is calculated, the stable nitrogen isotope value of the nitrate nitrogen assimilation product during the experiment treatment is obtained, the stable nitrogen isotope fractionation value of the woody plant assimilation nitrate nitrogen is calculated, and the nitrate supply-demand relationship of the woody plant is evaluated according to the stable nitrogen isotope fractionation value; the stable isotope value of the nitrate nitrogen assimilation product of the leaves, stems and roots is calculated according to an isotope mass balance model, the source of the newly added stem nitrogen is calculated according to a two-end element isotope mixing model, and the total amount of nitrogen assimilation of the leaves and roots is calculated. The application can quantitatively evaluate the nitrate supply-demand relationship and nitrate assimilation capacity of the woody plants, and fills the blank of precise fertilization under the condition that nitrate nitrogen is used as the only nitrogen source.

Description

Technical Field

[0001] The invention relates to a method for evaluating the nitrate supply-demand relationship of woody plants and the distribution of nitrate assimilation products, and belongs to the field of agricultural resource evaluation technology and biomass energy production technology. Background Art

[0002] Under well-aerated soil conditions or high pH (>7), such as in karst environments, nitrate nitrogen is the primary inorganic nitrogen form in the soil, and most plants absorb and utilize this inorganic nitrogen primarily as nitrate. Plant growth and development typically depend on the availability of inorganic nitrogen. Low nitrogen levels can inhibit plant growth and development, while excessive inorganic nitrogen supply not only wastes nitrogen fertilizer but also leads to environmental problems. Therefore, understanding the relationship between plant nitrate supply and demand is crucial for precise fertilization.

[0003] Stable nitrogen isotope fractionation values ​​can indicate the relationship between the external nitrogen supply and the plant's nitrogen demand. Generally, smaller nitrogen isotope fractionation values ​​indicate that the external nitrogen supply is closer to the plant's nitrogen demand (i.e., the inorganic nitrogen supply-demand balance), while larger nitrogen isotope fractionation values ​​indicate that the external nitrogen supply is lower than or higher than the plant's nitrogen demand. Therefore, quantifying the stable nitrogen isotope fractionation values ​​of woody plants at the whole-plant scale when nitrate nitrogen is the sole nitrogen source can estimate the nitrate supply-demand relationship of woody plants, thereby scientifically managing the inorganic nitrogen supply in karst environments.

[0004] Nitrogen assimilation in plants primarily occurs in leaves and roots. Therefore, organic nitrogen in woody plant stems primarily derives from the transfer of leaf nitrogen assimilation products and root nitrogen assimilation products. Quantifying the proportion of newly added organic nitrogen in the stem—that is, the proportion derived from leaf nitrogen assimilation products and the proportion derived from root nitrogen assimilation products—can estimate the amount of nitrate assimilation in leaves and roots of woody plants when nitrate nitrogen is the sole nitrogen source, and thus assess the contribution of leaves and roots to nitrate assimilation. Understanding the contribution of leaves and roots to nitrate assimilation in woody plants provides a deeper understanding of nitrogen metabolism in woody plants. However, current methods rely primarily on measuring nitrate reductase activity and nitrate assimilation capacity in roots, stems, and leaves to assess plant nitrate supply and demand and the distribution of nitrate assimilation products. This method is complex, inefficient, and inaccurate, making it difficult to achieve precise fertilization when nitrate nitrogen is the sole nitrogen source. Research and improvement are necessary. Summary of the Invention

[0005] Based on the above, the present invention provides a method for evaluating the nitrate supply and demand relationship and the distribution of nitrate assimilation products in woody plants, quantitatively evaluating the nitrate supply and demand relationship and nitrate assimilation capacity of woody plants, and filling the gap in the inability to accurately fertilize when nitrate nitrogen is the only nitrogen source.

[0006] The technical solution of the present invention is: a method for evaluating the nitrate supply and demand relationship and the distribution of nitrate assimilation products in woody plants, comprising the following steps:

[0007] First, woody plant seedlings with consistent growth were selected for the experiment. Before the experiment, the dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of the woody plant seedlings were measured. The initial dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves of the woody plant seedlings were recorded as DW, l0 、N l0 and δ 15 N l0 , the initial dry weight, nitrogen content and stable nitrogen isotope value of the stem were recorded as DW s0 、N s0 and δ 15 N s0 The initial dry weight, nitrogen content and stable nitrogen isotope value of the root were recorded as DW r0 、N r0 and δ 15 N r0 , using DW l0 、N l0 , δ 15 N l0 、DW s0 、N s0 , δ 15 N s0 、DW r0 、N r0 and δ 15 N r0 , calculate the initial stable nitrogen isotope value and total nitrogen accumulation of the whole woody plant, which are recorded as δ 15 N whole-plant0 , and m whole-plant0 ;

[0008] Among them, δ 15 N whole-plant0 The calculation equation is as follows:

[0009] δ 15 N whole-plant0 (‰)=(DW l0 ×N l0 ×δ 15 N l0 +DW s0 ×N s0 ×δ 15 N s0 +DW r0 ×N r0 ×δ 15 N r0 ) / (DW l0 ×N l0 +DW s0 ×Ns0 +DW r0 ×N r0 )

[0010] m whole-plant0 The calculation equation is as follows:

[0011] m whole-plant0 =DW l0 ×N l0 +DW s0 ×N s0 +DW r0 ×N r0 .

[0012] Second, the woody plant seedlings were planted in a nutrient solution containing only nitrate nitrogen. After a period of cultivation, the dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of the woody plant seedlings were measured. The dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves of the woody plant seedlings after the experimental treatment were recorded as DW, l1 、N l1 and δ 15 N l1 , the stem dry weight, nitrogen content and stable nitrogen isotope value were recorded as DW s1 、N s1 and δ 15 N s1 , the dry weight, nitrogen content and stable nitrogen isotope value of the root were recorded as DW r1 、N r1 and δ 15 N r1 , using DW l1 、N l1 , δ 15 N l1 、DW s1 、N s1 , δ 15 N s1 、DW r1 、N r1 and δ 15 N r1 , calculate the stable nitrogen isotope value and total nitrogen accumulation of the whole woody plant after the experimental treatment, and record them as δ 15 N whole-plant1 , and m whole-plant1 ;

[0013] Among them, δ 15 N whole-plant1 The calculation equation is as follows:

[0014] δ 15 N whole-plant1 (‰)=(DW l1 ×N l1 ×δ 15 Nl1 +DW s1 ×N s1 ×δ 15 N s1 +DW r1 ×N r1 ×δ 15 N r1 ) / (DW l1 ×N l1 +DW s1 ×N s1 +DW r1 ×N r1 );

[0015] m whole-plant1 The calculation equation is as follows:

[0016] m whole-plant1 =DW l1 ×N l1 +DW s1 ×N s1 +DW r1 ×N r1 .

[0017] Third, according to the isotope mass balance equation, based on δ 15 N whole-plant0 , δ 15 N whole-plant1 、m whole-plant0 and m whole-plant1 , calculate the stable isotope value of nitrogen assimilation products of the whole woody plant, recorded as δ 15 N assimilates ;

[0018] Among them, δ 15 N assimilates The calculation equation is as follows:

[0019] δ 15 N assimilates (‰)=(m whole-plant1 ×δ 15 N whole-plant1 -m whole-plant0 ×δ 15 N whole-plant0 ) / (m whole-plant1 -m whole-plant0 ).

[0020] Fourth, according to δ 15 N assimilates Calculate the stable nitrogen isotope fractionation value of the nitrogen assimilation products of the whole woody plant, denoted as Δ 15 N assimilates , and according to Δ 15 N assimilatesThe size of the nitrate supply and demand relationship of woody plants is assessed;

[0021] Among them, Δ 15 N assimilates The calculation equation is as follows:

[0022] Δ 15 N assimilates (‰)=δ 15 N substrate -δ 15 N assimilates

[0023] Where, δ 15 N substrate It is the stable nitrogen isotope value of nitrate nitrogen in chemical drugs.

[0024] Fifth, according to the isotope mass balance equation, using DW l0 、N l0 , δ 15 N l0 、DW l1 、N l1 and δ 15 N l1 , calculate the stable nitrogen isotope value of the leaf nitrogen assimilation product, recorded as δ 15 N leaf-assimilates , using DW s0 、N s0 , δ 15 N s0 、DW s1 、N s1 and δ 15 N s1 , calculate the stable nitrogen isotope value of the stem nitrogen assimilation product, denoted as δ 15 N stem-assimilates , using DW r0 、N r0 , δ 15 N r0 、DW r1 、N r1 and δ 15 N s1 , calculate the stable nitrogen isotope value of the nitrogen assimilation product of the root, recorded as δ 15 N root-assimilates ;

[0025] Among them, δ 15 N leaf-assimilates The calculation equation is as follows:

[0026] δ 15 N leaf-assimilates (‰)=(DW l1 ×N l1 ×δ 15 Nl1 -DW l0 ×N l0 ×δ 15 N l0 ) / (DW l1 ×N l1 -DW l0 ×N l0 )

[0027] δ 15 N stem-assimilates The calculation equation is as follows:

[0028] δ 15 N stem-assimilates (‰)=(DW s1 ×N s1 ×δ 15 N s1 -DW s0 ×N s0 ×δ 15 N s0 ) / (DW s1 ×N s1 -DW s0 ×N s0 )

[0029] δ 15 N root-assimilates The calculation equation is as follows:

[0030] δ 15 N root-assimilates (‰)=(DW r1 ×N r1 ×δ15N r1 -DW r0 ×N r0 ×δ15N r0 ) / (DW r1 ×N r1 -DW r0 ×N r0 ).

[0031] Sixth, according to the two-terminal isotope mixing model, using δ 15 N leaf-assimilates , δ 15 N stem-assimilates and δ 15 N root-assimilates Calculate the proportion of new stem nitrogen in woody plants that comes from leaf nitrogen assimilation, denoted as f leafstem ; The part derived from nitrogen assimilation in roots is denoted as 1–f leafstem ;

[0032] Among them, f leafstem The calculation equation is as follows:

[0033]

[0034] Seventh, according to f leafstem or 1–f leafstem ; Calculate the total amount of nitrogen assimilation products in the leaves and roots of woody plants, respectively, and record them as m leaf and m root , and then evaluate the contribution of leaves and roots to nitrate assimilation;

[0035] Among them, m leaf The calculation equation is as follows:

[0036] m leaf =(DW l1 ×N l1 -DW l0 ×N l0 )+f leaf stem ×(DW s1 ×N s1 -DW s0 ×N s0 )

[0037] m root The calculation equation is as follows:

[0038] m root =(DW r1 ×N r1 -DW r0 ×N r0 )+(1-f leaf stem )×(DW s1 ×N s1 -DW s0 ×N s0 ).

[0039] Eighth, according to m leaf and m root , calculate the nitrate assimilation contribution of woody plant leaves, recorded as P, and the nitrate assimilation contribution of woody plant roots is 1–P;

[0040] The calculation equation of P is as follows:

[0041]

[0042] The nutrient solution is configured with an appropriate nitrogen concentration using nitrate as the sole nitrogen source, and the stable nitrogen isotope value in the nitrate is greater than 20‰.

[0043] The beneficial effects of the present invention are: it can evaluate the nitrate supply and demand relationship of woody plants growing under conditions where nitrate nitrogen is the sole nitrogen source or in karst environments, providing a theoretical basis for scientific management of the nitrate nitrogen supply of woody plants. Furthermore, the present invention can estimate the total amount of nitrogen assimilated by the leaves and roots of woody plants, and further evaluate the contribution of nitrate nitrogen to nitrate assimilation by the leaves and roots of woody plants under conditions where nitrate nitrogen is the sole nitrogen source or in karst environments, providing a scientific basis for a deeper understanding of the inorganic nitrogen utilization strategies of karst-adapted plants and for precise fertilization. Compared with existing technologies, the present invention has the following advantages:

[0044] 1) The present invention only requires simple measurement of the biomass, nitrogen content, and stable nitrogen isotope value of the plant roots, stems, and leaves, without the need to measure the nitrate reductase activity and nitrate assimilation capacity of the plant roots, stems, and leaves, to obtain the plant nitrate supply and demand relationship and the distribution of nitrate assimilation products, and the steps are simple.

[0045] 2) The present invention can quantitatively evaluate the nitrate supply and demand relationship of woody plants under different nitrate nitrogen concentrations, providing a scientific basis for precise nitrogen fertilizer management.

[0046] 3) The present invention can quantitatively evaluate the distribution of nitrate assimilation products in plant leaves and roots under karst environments, which helps to deeply understand the inorganic nitrogen utilization strategies of karst-adapted plants and provides a theoretical basis for improving vegetation productivity in karst areas.

[0047] 4) The present invention is based on the isotope mass balance equation and the two-terminal isotope mixing model, and the calculation results are accurate.

[0048] The technical principle of the present invention is:

[0049] Stable nitrogen isotope technology has been widely used to study nitrogen metabolism in plants. The two stable nitrogen isotopes of nitrogen in nature are 14 N and 15 N, stable nitrogen isotope values ​​are usually expressed in δ 15 N (‰) indicates that plants usually experience nitrogen isotope fractionation when assimilating inorganic nitrogen, which leads to the δ 15 N value is less than the δ of the culture medium 15 N value. According to the δ 15 The N value can be used to calculate the stable nitrogen isotope fractionation value of inorganic nitrogen assimilation by plants, that is, Δ 15 N value. By Δ 15 The size of the N value can be used to determine the nitrate supply and demand relationship of the plant. The smaller the nitrogen isotope fractionation value, the closer the external nitrogen supply is to the nitrogen demand of the plant (that is, the supply and demand balance of inorganic nitrogen), and the larger the nitrogen isotope fractionation value, the lower or higher the external nitrogen supply is than the nitrogen demand of the plant.

[0050] Nitrate assimilation in woody plants typically occurs in leaves and roots. Therefore, evaluating the stable nitrogen isotope values ​​of nitrogen assimilation products in woody plants requires comprehensive consideration of the stable nitrogen isotope values ​​and nitrogen accumulation in the leaves, stems, and roots of woody plants before and after the experimental treatment. Specifically, the stable nitrogen isotope values ​​and nitrogen accumulation of the entire woody plant before and after the experimental treatment must be calculated. The stable nitrogen isotope values ​​of nitrogen assimilation products of the entire woody plant can then be calculated based on the isotope mass balance equation.

[0051] The stable nitrogen isotope values ​​of the whole woody plant were calculated using the following equation:

[0052] δ 15 N whole-plant0 (‰)=(DW l0 ×N l0 ×δ 15 N l0 +DW s0 ×N s0 ×δ 15 N s0 +DW r0 ×N r0 ×δ 15 N r0 ) / (DW l0 ×N l0 +DW s0 ×N s0 +DW r0 ×N r0 ) (1)

[0053] δ 15 N whole-plant1 (‰)=(DW l1 ×N l1 ×δ 15 N l1 +DW s1 ×N s1 ×δ 15 N s1 +DW r1 ×N r1 ×δ 15 N r1 ) / (DW l1 ×Ν l1 +DW s1 ×Ν s1 +DW r1 ×Ν r1 ) (2)

[0054] Here δ 15 N whole-plant0 and δ 15 N whole-plant1 are the stable nitrogen isotope values ​​of the whole woody plant before and after the experimental treatment; DWl0 and DW l1 are the dry weight of woody plant leaves before and after experimental treatment; DW s0 and DW s1 are the dry weight of woody plant stems before and after experimental treatment; DW r0 and DW r1 are the dry weight of woody plant roots before and after experimental treatment; N l0 and N l1 are the nitrogen content of woody plant leaves before and after experimental treatment; N s0 and N s1 are the nitrogen contents of woody plant stems before and after experimental treatment; N r0 and N r1 are the nitrogen contents of woody plant roots before and after experimental treatment; δ 15 N l0 and δ 15 N l1 are the stable nitrogen isotope values ​​of woody plant leaves before and after experimental treatment; δ 15 N s0 and δ 15 N s1 are the stable nitrogen isotope values ​​of woody plant stems before and after experimental treatment; δ 15 N r0 and δ 15 N r1 are the stable nitrogen isotope values ​​of woody plant roots before and after experimental treatment;

[0055] The total nitrogen accumulation of the whole woody plant was calculated using the following equation:

[0056] m whole-plant0 =DW l0 ×Ν l0 +DW s0 ×Ν s0 +DW r0 ×N r0 (3)

[0057] m whole-plant1 =DW l1 ×N l1 +DW s1 ×Ν s1 +DW r1 ×Ν r1 (4)

[0058] According to the isotope mass balance equation, using δ 15 N whole-plant0 , δ 15 N whole-plant1 、mwhole-plant0 and m whole-plant1 The stable isotope value of nitrogen assimilation products of the whole woody plant (δ 15 N assimilates ):

[0059] δ 15 N assimilates (‰)=(m whole-plant1 ×δ 15 N whole-plant1 -m whole-plant0 ×δ 15 N whole-plant0 ) / (m whole-plant1 -m whole-plant0 ) (5)

[0060] Based on the stable isotope value of nitrogen assimilation products of the whole woody plant, the stable nitrogen isotope fractionation value of nitrogen assimilation products of the whole woody plant (Δ 15 N assimilates ):

[0061] Δ 15 N assimilates (‰)=δ 15 N substrate -δ 15 N assimilates (6)

[0062] Here δ 15 N substrate is the stable nitrogen isotope value of nitrate nitrogen in chemical drugs;

[0063] The organic nitrogen in the stems of woody plants mainly comes from the transfer of nitrogen assimilation products from leaves and roots. Therefore, the stable nitrogen isotope value of the newly added organic nitrogen in the stems of woody plants is the result of the mixing of the stable nitrogen isotope values ​​of the nitrogen assimilation products of leaves and roots. Accordingly, using the two-terminal isotope mixing model, the source ratio of the newly added organic nitrogen in woody plants can be calculated, that is, the ratio from nitrogen assimilation products in leaves and the ratio from nitrogen assimilation products in roots. The ratio from nitrogen assimilation in leaves is recorded as f leafstem ; The portion derived from nitrogen assimilation in roots is recorded as 1–f leafstem ;

[0064] The isotope mixing model of the two terminal members is expressed as:

[0065] δ 15 N stem-assimilates =f leaf stem ×δ 15 N leaf-assimilates +(1-f leaf stem )×δ 15 N root-assimilates (7)

[0066] Here δ 15 N leaf-assimilates is the stable nitrogen isotope value of leaf nitrogen assimilation products, δ 15 N root-assimilates is the stable nitrogen isotope value of nitrogen assimilation products in roots, δ 15 N stem-assimilates Stable nitrogen isotope values ​​of organic nitrogen in the stems (stable nitrogen isotope values ​​of nitrogen assimilation products in leaves and roots mixed together) were added.

[0067] According to the stable nitrogen isotope values ​​and total nitrogen accumulation of woody plant leaves, stems and roots before and after the experimental treatment, the isotope mass balance equation can be used to calculate δ 15 N leaf-assimilates , δ 15 N stem-assimilates and δ 15 N root-assimilates ;

[0068] The isotope mass balance equation is expressed as:

[0069] δ 15 N leaf-assimilates (‰)=(DW l1 ×N l1 ×δ 15 N l1 -DW l0 ×N l0 ×δ 15 N l0 ) / (DW l1 ×N l1 -DW l0 ×N l0 ) (8)

[0070] δ 15 N stem-assimilates (‰)=(DW s1 ×N s1 ×δ 15 N s1 -DW s0 ×N s0 ×δ 15 N s0 ) / (DW s1 ×N s1 -DW s0 ×N s0 ) (9)

[0071] δ 15 N root-assimilates (‰)=(DW r1 ×N r1 ×δ 15 N r1 -DWr0 ×N r0 ×δ 15 N r0 ) / (DW r1 ×N r1 -DW r0 ×N r0 ) (10)

[0072] Calculate δ 15 N leaf-assimilates , δ 15 N stem-assimilates and δ 15 N root-assimilates Then, equation (7) is transformed into the following equation:

[0073]

[0074] According to equation (11), the proportion of new stem nitrogen of woody plants that comes from nitrogen assimilation products of leaves can be calculated. Correspondingly, the proportion of new stem nitrogen of woody plants that comes from nitrogen assimilation products of roots is 1 – f leafstem The total amount of nitrogen assimilation products in the leaves and roots of woody plants can be obtained by solving the following equation:

[0075] m leaf =(DW l1 ×N l1 -DW l0 ×N l0 )+f leaf stem ×(DW s1 ×N s1 -DW s0 ×N s0 ) (12)

[0076] m root =(DW r1 ×N r1 -DW r0 ×N r0 )+(1-f leaf stem )×(DW s1 ×N s1 -DW s0 ×N s0 )(13)

[0077] The m here leaf is the total amount of nitrogen assimilation products in woody plant leaves, m root is the total amount of nitrogen assimilation products in the roots of woody plants;

[0078] According to m leaf and m root , the nitrate assimilation contribution (P) of woody plant leaves can be calculated by the following equation:

[0079]

[0080] Correspondingly, the contribution of nitrate assimilation by the roots of woody plants is 1–P. DETAILED DESCRIPTION

[0081] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0082] The embodiment of the present invention comprises the following steps:

[0083] First, the stable nitrogen isotope ratio of the nitrate used as the sole nitrogen source in the nutrient solution is measured using a stable isotope mass spectrometer to screen for nitrates with a stable nitrogen isotope ratio greater than 20‰. This nitrate is then used as the sole nitrogen source to prepare a nutrient solution with an appropriate nitrogen concentration. In this example, the nitrate nitrogen selected was sodium nitrate with the same stable nitrogen isotope composition from the same manufacturer and batch.

[0084] Second, all cultures were selected to be cultured in the same culture room.

[0085] Third, cultivating woody plant seedlings of uniform growth in the above nutrient solution;

[0086] Fourth, similarly, three woody plant seedlings with the same growth were randomly selected, and the dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of these three seedlings were measured. The average dry weight, average leaf nitrogen content, and average stable nitrogen isotope value of the leaves, stems, and roots of these three seedlings were approximately the initial dry weight, initial nitrogen content, and initial stable nitrogen isotope value of the leaves, stems, and roots of the woody plant seedlings in the entire experimental treatment; the initial dry weight, nitrogen content, and stable nitrogen isotope value of the leaves of the woody plant seedlings were recorded as DW, l0 、N l0 and δ 15 N l0 ; The initial dry weight, nitrogen content and stable nitrogen isotope value of the stem were recorded as DW s0 、N s0 and δ 15 N s0 The initial dry weight, nitrogen content and stable nitrogen isotope value of the root were recorded as DW r0 、N r0 and δ 15 N r0 ;

[0087] Fifth, after 20 days of culture, the treatment solution was replaced every 2 days, with 500 ml of treatment solution per seedling. The dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of the woody plants were measured. The dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves of the woody plant seedlings 20 days after treatment were recorded as DW, l1 、N l1 and δ 15 N l1 ; Stem dry weight, nitrogen content and stable nitrogen isotope value were recorded as DW s1 、N s1 and δ 15 N s1 ; The dry weight, nitrogen content and stable nitrogen isotope value of the root were recorded as DW r1 、N r1 and δ 15 N r1 ;

[0088] Sixth, use DW l0 、N l0 , δ 15 N l0 、DW s0 、N s0 , δ 15 N s0 、DW r0 、N r0 and δ 15 N r0 , calculate the initial stable nitrogen isotope value and total nitrogen accumulation of the whole woody plant; denoted as δ 15 N whole-plant0 , and m whole-plant0 The method for calculating the stable nitrogen isotope value of the whole woody plant is as follows: DW l0 、N l0 , δ 15 N l0 、DW s0 、N s0 , δ 15 N s0 、DW r0 、N r0 and δ 15 N r0 Substituting into the equation: δ 15 N whole-plant0 (‰)=(DW l0 ×N l0 ×δ 15 N l0 +DW s0 ×N s0 ×δ 15 N s0 +DW r0 ×N r0×δ 15 N r0 ) / (DW l0 ×Ν l0 +DW s0 ×Ν s0 +DW r0 ×Ν r0 ); the method for calculating the total nitrogen accumulation of the whole woody plant is: DW l0 、N l0 、DW s0 、N s0 、DW r0 and N r0 Substitute into the equation: m whole-plant0 =DW l0 ×Ν l0 +DW s0 ×Ν s0 +DW r0 ×Ν r0 ;

[0089] Seventh, use DW l1 、N l1 , δ 15 N l1 、DW s1 、N s1 , δ 15 N s1 、DW r1 、N r1 and δ 15 N r1 , calculate the stable nitrogen isotope value and total nitrogen accumulation of the whole woody plant after 20 days of cultivation; record it as δ 15 N whole-plant1 , and m whole-plant1 The method for calculating the stable nitrogen isotope value of the whole woody plant is as follows: DW l1 、N l1 , δ 15 N l1 、DW s1 、N s1 , δ 15 N s1 、DW r1 、N r1 and δ 15 N r1 Substituting into the equation: δ 15 N whole-plant1 (‰)=(DW l1 ×N l1 ×δ 15 N l1 +DW s1 ×N s1 ×δ 15 N s1 +DWr1 ×N r1 ×δ 15 N r1 ) / (DW l1 ×Ν l1 +DW s1 ×Ν s1 +DW r1 ×Ν r1 ); The method for calculating the total nitrogen accumulation of the whole woody plant is: m whole-plant1 =DW l1 ×Ν l1 +DW s1 ×Ν s1 +DW r1 ×Ν r1 ;

[0090] Eighth, according to the isotope mass balance equation, based on δ 15 N whole-plant0 , δ 15 N whole-plant1 、m whole-plant0 and m whole-plant1 The stable isotope value of nitrogen assimilation products of the whole woody plant can be calculated; it is recorded as δ 15 N assimilates The method for calculating the stable isotope value of nitrogen assimilation products of the whole woody plant is: δ 15 N assimilates (‰)=(m whole-plant1 ×δ 15 N whole-plant1 -m whole-plant0 ×δ 15 N whole-plant0 ) / (m whole-plant1 -m whole-plant0 );

[0091] Ninth, according to δ 15 N assimilates Calculate the stable nitrogen isotope fractionation value of the nitrogen assimilation products of the whole woody plant, denoted as Δ 15 N assimilates The method for calculating the stable nitrogen isotope fractionation value of the nitrogen assimilation products of the whole woody plant is as follows: 15 N assimilates Substituting into the equation: Δ 15 N assimilates (‰)=δ 15 N substrate -δ 15 N assimilates , δ 15 N substrate is the stable nitrogen isotope value of nitrate nitrogen in chemical drugs;

[0092] Tenth, according to Δ 15 Nassimilates The size of the nitrate supply and demand relationship of woody plants is assessed;

[0093] Eleventh, according to the isotope mass balance equation, using DW l0 、N l0 , δ 15 N l0 、DW l1 、N l1 and δ 15 N l1 , calculate the stable nitrogen isotope value of the leaf nitrogen assimilation product, recorded as δ 15 N leaf-assimilates The method for calculating the stable nitrogen isotope value of the nitrogen assimilation product of the leaf is as follows: DW l0 、N l0 , δ 15 N l0 、DW l1 、N l1 and δ 15 N l1 Substituting into the equation: δ 15 N leaf-assimilates (‰)=(DW l1 ×N l1 ×δ 15 N l1 -DW l0 ×N l0 ×δ 15 N l0 ) / (DW l1 ×N l1 -DW l0 ×N l0 );

[0094] 12. According to the isotope mass balance equation, using DW s0 、N s0 , δ 15 N s0 、DW s1 、N s1 and δ 15 N s1 , calculate the stable nitrogen isotope value of the stem nitrogen assimilation product, denoted as δ 15 N stem-assimilates ; The method for calculating the stable nitrogen isotope value of stem nitrogen assimilation products is: DW s0 、N s0 , δ 15 N s0 、DW s1 、N s1 and δ 15 N s1 Substituting into the equation: δ 15 N stem-assimilates(‰)=(DW s1 ×N s1 ×δ 15 N s1 -DW s0 ×N s0 ×δ 15 N s0 ) / (DW s1 ×N s1 -DW s0 ×N s0 );

[0095] Thirteenth, according to the isotope mass balance equation, using DW r0 、N r0 , δ 15 N r0 、DW r1 、N r1 and δ 15 N s1 , calculate the stable nitrogen isotope value of the nitrogen assimilation product of the root, recorded as δ 15 N root-assimilates ; The method for calculating the stable nitrogen isotope value of nitrogen assimilation products of roots is: DW r0 、N r0 , δ 15 N r0 、DW r1 、N r1 and δ 15 N r1 Substituting into the equation: δ 15 N root-assimilates (‰)=(DW r1 ×N r1 ×δ 15 N r1 -DW r0 ×N r0 ×δ 15 N r0 ) / (DW r1 ×N r1 -DW r0 ×N r0 );

[0096] Fourteenth, according to the two-terminal isotope mixing model, using δ 15 N leaf-assimilates , δ 15 N stem-assimilates and δ 15 N root-assimilates Calculate the proportion of new stem nitrogen in woody plants that comes from leaf nitrogen assimilation, denoted as f leafstem ; The part derived from nitrogen assimilation in roots is denoted as 1–f leafstem The method for calculating the proportion of new stem nitrogen in woody plants that comes from leaf nitrogen assimilation is as follows:15 N leaf-assimilates , δ 15 N stem-assimilates and δ 15 N root-assimilates Substituting into the equation:

[0097] Fifteenth, according to f leafstem or 1–f leafstem ; Calculate the total amount of nitrogen assimilation products in the leaves and roots of woody plants, respectively, and record them as m leaf and m root , and then evaluate the contribution of leaves and roots to nitrate assimilation; the method for calculating the total amount of nitrogen assimilation products in leaves and roots of woody plants is: DW l0 、N l0 、DW l1 、N l1 、DW s0 、N s0 、DW s1 、N s1 and f leafstem Substitute into the equation: m leaf =(DW l1 ×N l1 -DW l0 ×N l0 )+f leaf stem ×(DW s1 ×N s1 -DW s0 ×N s0 ); DW r0 、N r0 、DW r1 、N r1 、DW s0 、N s0 、DW s1 、N s1 , and 1–f leafstem Substitute into the equation: m root =(DW r1 ×N r1 -DW r0 ×N r0 )+(1-f leaf stem )×(DW s1 ×N s1 -DW s0 ×N s0 );

[0098] Sixteenth, according to m leaf and m root Calculate the contribution of nitrate assimilation of woody plant leaves, denoted as P. The method for calculating the contribution of nitrate assimilation of woody plant leaves is: leaf and mroot Substituting into the equation: The contribution of nitrate assimilation by woody plant roots is 1–P;

[0099] Example 1:

[0100] Culture material: Paper mulberry seedlings with uniform growth

[0101] Culture conditions: Broussonetia papyrifera seedlings were cultured in an improved 1 / 2 Hoagland nutrient solution. Nitrate nitrogen was the only nitrogen source in the Hoagland nutrient solution, and its stable nitrogen isotope value was: δ 15 N substrate =22.35‰. The light intensity in the culture room was 500±25μmolm -2 s -1 , the temperature during light was 25±2℃, the temperature at night was 19±2℃, the relative humidity was 55-60%, and the pH value of the culture solution was 7.5±0.1. Since the nitrate nitrogen content in the soil of the karst area is much less than 10mM, the nitrate nitrogen concentrations in the Hogland nutrient solution were set to 0.5mM, 2mM and 8mM respectively. The paper mulberry seedlings were cultured in the above-mentioned Hogland nutrient solution for 20 days, wherein the above-mentioned culture solution was replaced every 2 days, and 500mL of the above-mentioned culture solution was replaced for each paper mulberry seedling each time; at the beginning of the experimental treatment, 3 paper mulberry seedlings with the same growth were randomly selected, and the dry weight, nitrogen content and stable nitrogen isotope values ​​of the leaves, stems and roots of these three seedlings were measured respectively. The average dry weight, average nitrogen content and average stable nitrogen isotope value of the leaves, stems and roots of these 3 seedlings are approximately the initial dry weight, initial nitrogen content and initial stable nitrogen isotope value of the leaves, stems and roots of the paper mulberry seedlings in the entire experimental treatment. The measured initial dry weight of the leaves DW l0 The initial dry weight of the stem was 0.348 g (n=3). s0 The initial dry weight of the root was 0.075 g (n=3). r0 The initial nitrogen content of the leaves was 0.070 g (n=3). l0 The initial nitrogen content of the stem was 4.53% (n=3). s0 The initial nitrogen content of the roots was 2.81% (n=3). r0 The initial stable nitrogen isotope value of leaves was 3.15% (n=3). 15 N l0 The initial stable nitrogen isotope value of the stem was 7.51‰ (n=3). 15 N s0 The initial stable nitrogen isotope value of the roots was 6.97‰ (n=3). 15 N r0The nitrate nitrogen content of the paper mulberry seedlings was 6.46‰ (n=3). After the paper mulberry seedlings were cultured at the three nitrate nitrogen concentrations for 20 days, the dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of the paper mulberry seedlings were measured. The results are shown in Table 1:

[0102] Table 1 Dry weight, nitrogen content and stable nitrogen isotope values ​​of leaves, stems and roots of paper mulberry seedlings under nitrate nitrogen treatment

[0103]

[0104]

[0105] Note: n=3. DW l1 is the dry weight of the leaf, DW s1 is the dry weight of the stem, DW r1 is the dry weight of the root, N l1 is the nitrogen content of the leaves, N s1 is the nitrogen content of the stem, N r1 is the nitrogen content of the roots, δ 15 N l1 is the stable nitrogen isotope value of leaves, δ 15 N s1 is the stable nitrogen isotope value of the stem, δ 15 N r1 is the stable nitrogen isotope value of the root.

[0106] As shown in Table 1, increasing the concentration of nitrate nitrogen helps promote the growth and nitrogen assimilation of paper mulberry seedlings. In addition, the stable nitrogen isotope values ​​of paper mulberry seedlings leaves, stems and roots gradually increase with the increase of nitrate nitrogen concentration. According to the data in Table 1, combined with the initial dry weight, initial nitrogen content and initial stable nitrogen isotope values ​​of paper mulberry seedlings leaves, stems and roots, using equation δ 15 N whole-plant0 (‰)=(DW l0 ×N l0 ×δ 15 N l0 +DW s0 ×N s0 ×δ 15 N s0 +DW r0 ×N r0 ×δ 15 N r0 ) / (DW l0 ×Ν l0 +DW s0 ×Ν s0 +DW r0 ×Ν r0 ) and δ 15 N whole-plant1 (‰)=(DW l1 ×N l1 ×δ15 N l1 +DW s1 ×N s1 ×δ 15 N s1 +DW r1 ×N r1 ×δ 15 N r1 ) / (DW l1 ×Ν l1 +DW s1 ×Ν s1 +DW r1 ×Ν r1 ) can be used to calculate the stable nitrogen isotope values ​​of the whole paper mulberry seedling before and after the experimental treatment; using equation m whole-plant0 =DW l0 ×Ν l0 +DW s0 ×Ν s0 +DW r0 ×Ν r0 and m whole-plant1 =DW l1 ×Ν l1 +DW s1 ×Ν s1 +DW r1 ×Ν r1 The total nitrogen accumulation of the whole paper mulberry seedling before and after the experimental treatment can be calculated; finally, according to the equation δ 15 N assimilates (‰)=(m whole-plant1 ×δ 15 N whole-plant1 -m whole-plant0 ×δ 15 N whole-plant0 ) / (m whole-plant1 -m whole-plant0 ) and Δ 15 N assimilates (‰)=δ 15 N substrate -δ 15 N assimilates The stable nitrogen isotope value (δ 15 N assimilates ) and nitrogen isotope fractionation values ​​(Δ 15 N assimilates ), δ 15 N assimilates and Δ 15 N assimilates The calculation results are shown in Table 2:

[0107] Table 2 Stable nitrogen isotope values ​​and stable nitrogen isotope fractionation values ​​of nitrogen assimilation products of whole Broussonetia papyrifera seedlings under nitrate nitrogen treatment

[0108]

[0109] Note: The stable nitrogen isotope value of nitrate nitrogen in chemical drugs is: δ 15 N substrate =22.35‰.

[0110] As shown in Table 2, with the increase of nitrate nitrogen concentration, the stable nitrogen isotope value of the nitrogen assimilation product of paper mulberry seedlings gradually increased, that is, increasing the nitrate nitrogen concentration is conducive to the enrichment of paper mulberry seedlings. 15 N. When nitrate nitrogen concentrations ranged from 0.5 to 8 mM, the nitrogen isotope fractionation of the nitrogen assimilation products of paper mulberry seedlings gradually decreased with increasing nitrate nitrogen concentrations. The nitrogen isotope fractionation of the nitrogen assimilation products of paper mulberry seedlings reached its minimum at 8 mM nitrate nitrogen concentration. Therefore, at a nitrate nitrogen concentration of 8 mM, the inorganic nitrogen supply was close to the inorganic nitrogen requirement of the paper mulberry seedlings. This suggests that paper mulberry seedlings have a high demand for nitrate nitrogen, and a supply of 8 mM nitrate nitrogen does not exceed their inorganic nitrogen requirement. The nitrogen isotope fractionation of the nitrogen assimilation products of paper mulberry seedlings reached its maximum at a nitrate nitrogen concentration of 0.5 mM and was significantly greater than the nitrogen isotope fractionation at 8 mM, indicating that a 0.5 mM nitrate nitrogen supply was far below the inorganic nitrogen requirement of the paper mulberry seedlings. Based on the stable nitrogen isotope fractionation values ​​of nitrogen assimilation products of paper mulberry seedlings, the nitrate supply and demand relationship of paper mulberry seedlings under different nitrate nitrogen concentrations can be estimated, thereby avoiding the phenomenon of insufficient or excessive supply of inorganic nitrogen.

[0111] In addition, according to the dry weight, nitrogen content and stable nitrogen isotope values ​​of leaves, stems and roots of paper mulberry seedlings before and after experimental treatment, the isotope mass balance equation was used to calculate the nitrogen content of the stable nitrogen isotope. 15 N leaf-assimilates (‰)=(DW l1 ×N l1 ×δ 15 N l1 -DW l0 ×N l0 ×δ 15 N l0 ) / (DW l1 ×N l1 -DW l0 ×N l0 ),δ 15 N stem-assimilates (‰)=(DW s1 ×N s1 ×δ 15 N s1 -DW s0 ×N s0 ×δ 15 N s0 ) / (DWs1 ×N s1 -DW s0 ×N s0 ) and δ 15 N root-assimilates (‰)=(DW r1 ×N r1 ×δ 15 N r1 -DW r0 ×N r0 ×δ 15 N r0 ) / (DW r1 ×N r1 -DW r0 ×N r0 ) can be used to calculate the stable nitrogen isotope value (δ 15 N leaf-assimilates ), stable nitrogen isotope values ​​of root nitrogen assimilation products (δ 15 N root-assimilates ) and the stable nitrogen isotope values ​​of newly added organic nitrogen in the stems (δ 15 N stem-assimilates ); According to the two-terminal isotope mixing model, using equation The proportion of the newly added stem nitrogen of paper mulberry seedlings from the nitrogen assimilation products of leaves (f leafstem ), the proportion of newly added stem nitrogen of paper mulberry seedlings derived from nitrogen assimilation products in the roots is 1–f leafstem ;f leafstem and 1–f leafstem The calculation results are shown in Table 3:

[0112] Table 3 The proportion of newly added stem nitrogen in the whole Broussonetia papyrifera seedlings derived from leaf nitrogen assimilation products and the proportion derived from root nitrogen assimilation products under nitrate nitrogen treatment

[0113]

[0114] As shown in Table 3, with increasing nitrate nitrogen concentration, the proportion of new organic nitrogen in the stems of paper mulberry seedlings that originated from leaf nitrogen assimilation products gradually increased. At a nitrate nitrogen concentration of 0.5 mM, the proportion of new organic nitrogen in the stems that originated from leaf nitrogen assimilation products was only 0.1684, indicating that the new organic nitrogen in the stems primarily originated from the transfer of nitrogen assimilation products from the roots. When the nitrate nitrogen concentration increased to 8 mM, the proportion of new organic nitrogen in the stems that originated from leaf nitrogen assimilation products reached 0.5902, indicating that the new organic nitrogen in the stems primarily originated from the transfer of nitrogen assimilation products from the leaves.

[0115] According to f in Table 3 leafstem and 1–f leafstemCombining the biomass and nitrogen content of the roots, stems and leaves of the paper mulberry seedlings before and after the experimental treatment, the total nitrogen assimilation of the leaves and roots of the paper mulberry seedlings during the entire experimental treatment period can be calculated, see Table 4.

[0116] Table 4 Total nitrogen assimilation in leaves and roots of whole Broussonetia papyrifera seedlings under nitrate nitrogen treatment

[0117]

[0118] According to the total nitrogen assimilation in the leaves and roots of paper mulberry seedlings at different nitrate nitrogen concentrations in Table 4, the contribution of nitrate assimilation in the leaves and roots of paper mulberry seedlings at different nitrate nitrogen concentrations can be calculated, see Table 5.

[0119] Table 5 Nitrate assimilation contribution of leaves and roots of whole Broussonetia papyrifera seedlings under nitrate nitrogen treatment

[0120]

[0121] As shown in Table 5, the contribution of nitrate assimilation by leaves gradually increased with increasing nitrate nitrogen concentration. At the highest nitrate nitrogen concentration, the contribution of nitrate assimilation by leaves was more than twice that of roots. The contribution of nitrate assimilation by roots gradually decreased with increasing nitrate nitrogen concentration. In general, at low nitrate nitrogen concentrations, nitrate assimilation in the roots of paper mulberry seedlings predominated; whereas at high nitrate nitrogen concentrations, nitrate assimilation in the leaves primarily occurred. Therefore, using stable nitrogen isotope technology, it is possible to quantify the contribution of nitrate assimilation by leaves and roots in paper mulberry seedlings at different nitrate nitrogen concentrations, thereby providing a deeper understanding of nitrogen metabolism in paper mulberry seedlings.

[0122] Example 2:

[0123] Culture material: Mulberry seedlings with uniform growth

[0124] Culture conditions: Mulberry seedlings were cultured in an improved 1 / 2 Hoagland nutrient solution. Nitrate nitrogen was the only nitrogen source in the Hoagland nutrient solution, and its stable nitrogen isotope value was: δ 15 N substrate =22.35‰. The light intensity in the culture room was 500±25μmolm -2 s -1, the temperature during light was 25±2℃, the temperature at night was 19±2℃, the relative humidity was 55-60%, and the pH value of the culture solution was 7.5±0.1. Since the nitrate nitrogen content in the soil of the karst area is much less than 10mM, the nitrate nitrogen concentrations in the Hogland nutrient solution were set to 0.5mM, 2mM and 8mM respectively. The mulberry seedlings were cultured in the above-mentioned Hogland nutrient solution for 20 days, wherein the above-mentioned culture solution was replaced every 2 days, and 500mL of the above-mentioned culture solution was replaced for each mulberry seedling each time; at the beginning of the experimental treatment, 3 mulberry seedlings with the same growth were randomly selected, and the dry weight, nitrogen content and stable nitrogen isotope values ​​of the leaves, stems and roots of these three seedlings were measured respectively. The average dry weight, average nitrogen content and average stable nitrogen isotope value of the leaves, stems and roots of these 3 seedlings are approximately the initial dry weight, initial nitrogen content and initial stable nitrogen isotope value of the leaves, stems and roots of the mulberry seedlings in the entire experimental treatment. The measured initial dry weight of the leaves DW l0 The initial dry weight of the stem was 0.207 g (n=3). s0 The initial dry weight of the root was 0.104 g (n=3). r0 The initial nitrogen content of the leaves was 0.079 g (n=3). l0 The initial nitrogen content of the stem was 4.09% (n=3). s0 The initial nitrogen content of the roots was 1.87% (n=3). r0 The initial stable nitrogen isotope value of leaves was 2.56% (n=3). 15 N l0 The initial stable nitrogen isotope value of the stem was 6.87‰ (n=3). 15 N s0 The initial stable nitrogen isotope value of the roots was 5.63‰ (n=3). 15 N r0 The nitrate nitrogen concentration in the mulberry seedlings was 4.95‰ (n=3). After the mulberry seedlings were cultured at the three nitrate nitrogen concentrations for 20 days, the dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of the mulberry seedlings were measured. The results are shown in Table 6:

[0125] Table 6 Dry weight, nitrogen content and stable nitrogen isotope values ​​of leaves, stems and roots of mulberry seedlings under nitrate nitrogen treatment

[0126]

[0127] Note: n=3. DW l1 is the dry weight of the leaf, DW s1 is the dry weight of the stem, DW r1 is the dry weight of the root, N l1 is the nitrogen content of the leaves, N s1 is the nitrogen content of the stem, N r1 is the nitrogen content of the roots, δ15 N l1 is the stable nitrogen isotope value of leaves, δ 15 N s1 is the stable nitrogen isotope value of the stem, δ 15 N r1 is the stable nitrogen isotope value of the root.

[0128] As shown in Table 6, when the nitrate nitrogen concentration was 0.5-2 mM, increasing the nitrate nitrogen concentration significantly promoted the growth of mulberry seedlings; however, when the nitrate nitrogen concentration exceeded 2 mM, continuing to increase the nitrate nitrogen concentration had a limited effect on the growth promotion of mulberry seedlings. In addition, the nitrogen content and stable nitrogen isotope values ​​of the leaves, stems and roots of mulberry seedlings gradually increased with the increase of nitrate nitrogen concentration. According to the data in Table 6, combined with the initial dry weight, initial nitrogen content and initial stable nitrogen isotope values ​​of the leaves, stems and roots of mulberry seedlings, using equation δ 15 N whole-plant0 (‰)=(DW l0 ×N l0 ×δ 15 N l0 +DW s0 ×N s0 ×δ 15 N s0 +DW r0 ×N r0 ×δ 15 N r0 ) / (DW l0 ×Ν l0 +DW s0 ×Ν s0 +DW r0 ×Ν r0 ) and δ 15 N whole-plant1 (‰)=(DW l1 ×N l1 ×δ 15 N l1 +DW s1 ×N s1 ×δ 15 N s1 +DW r1 ×N r1 ×δ 15 N r1 ) / (DW l1 ×Ν l1 +DW s1 ×N s1 +DW r1 ×N r1 ) can calculate the stable nitrogen isotope values ​​of the whole mulberry seedling before and after the experimental treatment; using equation m whole-plant0 =DW l0 ×Nl0 +DW s0 ×N s0 +DW r0 ×N r0 and m whole-plant1 =DW l1 ×N l1 +DW s1 ×N s1 +DW r1 ×N r1 The total nitrogen accumulation of the whole mulberry seedling before and after the experimental treatment can be calculated; finally, according to the equation δ 15 N assimilates (‰)=(m whole-plant1 ×δ 15 N whole-plant1 -m whole-plant0 ×δ 15 N whole-plant0 ) / (m whole-plant1 -m whole-plant0 ) and Δ 15 N assimilates (‰)=δ 15 N substrate -δ 15 N assimilates The stable nitrogen isotope value (δ 15 N assimilates ) and nitrogen isotope fractionation values ​​(Δ 15 N assimilates ), δ 15 N assimilates and Δ 15 N assimilates The calculation results are shown in Table 7:

[0129] Table 7 Stable nitrogen isotope values ​​and stable nitrogen isotope fractionation values ​​of nitrogen assimilation products of whole mulberry seedlings under nitrate nitrogen treatment

[0130]

[0131] Table 7 shows that with increasing nitrate nitrogen concentration, the stable nitrogen isotope values ​​of nitrogen assimilation products in mulberry seedlings initially increase and then decrease, reaching a maximum at 2 mM nitrate nitrogen and then decreasing at 8 mM nitrate nitrogen. This indicates that increasing nitrate nitrogen concentration does not simultaneously increase the stable nitrogen isotope values ​​of nitrogen assimilation products in mulberry seedlings. Within the nitrate nitrogen concentration range of 0.5 to 8 mM, the stable nitrogen isotope fractionation values ​​of nitrogen assimilation products in mulberry seedlings show a pattern of first decreasing significantly and then slowly increasing with increasing nitrate nitrogen concentration. The nitrogen isotope fractionation values ​​of nitrogen assimilation products in mulberry seedlings reach a minimum at 2 mM nitrate nitrogen concentration. Therefore, at a nitrate nitrogen concentration of 2 mM, the inorganic nitrogen supply is close to the inorganic nitrogen requirement of the mulberry seedlings. This indicates that the inorganic nitrogen requirement of mulberry seedlings is not very high. The nitrogen isotope fractionation values ​​of nitrogen assimilation products of mulberry seedlings at 8 mM nitrate nitrogen concentration were slightly higher than those at 2 mM nitrate nitrogen concentration, indicating that the 8 mM nitrate nitrogen supply exceeded the inorganic nitrogen demand of the mulberry seedlings. The nitrogen isotope fractionation values ​​of nitrogen assimilation products of mulberry seedlings reached a maximum value at 0.5 mM nitrate nitrogen concentration and were significantly higher than those at 2 mM, indicating that the 0.5 mM nitrate nitrogen supply was far lower than the inorganic nitrogen demand of the mulberry seedlings. Based on the stable nitrogen isotope fractionation values ​​of nitrogen assimilation products of mulberry seedlings, the nitrate supply and demand relationship of mulberry seedlings at different nitrate nitrogen concentrations can be estimated, thus avoiding the phenomenon of insufficient or excessive inorganic nitrogen supply.

[0132] In addition, according to the dry weight, nitrogen content and stable nitrogen isotope values ​​of leaves, stems and roots of mulberry seedlings before and after experimental treatment, the isotope mass balance equation was used to calculate the nitrogen content of the mulberry seedlings using the equation δ 15 N leaf-assimilates (‰)=(DW l1 ×N l1 ×δ 15 N l1 -DW l0 ×N l0 ×δ 15 N l0 ) / (DW l1 ×N l1 -DW l0 ×N l0 ),δ 15 N stem-assimilates (‰)=(DW s1 ×N s1 ×δ 15 N s1 -DW s0 ×N s0 ×δ 15 N s0 ) / (DW s1 ×N s1 -DWs0 ×N s0 ) and δ 15 N root-assimilates (‰)=(DW r1 ×N r1 ×δ 15 N r1 -DW r0 ×N r0 ×δ 15 N r0 ) / (DW r1 ×N r1 -DW r0 ×N r0 ) can be used to calculate the stable nitrogen isotope value (δ 15 N leaf-assimilates ), stable nitrogen isotope values ​​of root nitrogen assimilation products (δ 15 N root-assimilates ) and the stable nitrogen isotope values ​​of newly added organic nitrogen in the stems (δ 15 N stem-assimilates ); According to the two-terminal isotope mixing model, using equation The proportion of the newly added stem nitrogen of mulberry seedlings derived from the nitrogen assimilation products of leaves (f leafstem ), the proportion of new stem nitrogen of mulberry seedlings derived from nitrogen assimilation products in the roots is 1–f leafstem ;f leafstem and 1–f leafstem The calculation results are shown in Table 8:

[0133] Table 8 The proportion of newly added stem nitrogen in mulberry seedlings derived from leaf nitrogen assimilation products and the proportion derived from root nitrogen assimilation products under nitrate nitrogen treatment

[0134]

[0135] Table 8 shows that with increasing nitrate nitrogen concentration, the proportion of newly added organic nitrogen in the stems of mulberry seedlings originating from leaf nitrogen assimilation products initially increased and then decreased. At nitrate nitrogen concentrations of 0.5 mM and 8 mM, newly added organic nitrogen in the stems of mulberry seedlings primarily originated from transfer of root nitrogen assimilation products. At nitrate nitrogen concentrations of 2 mM, the proportion of newly added organic nitrogen in the stems originating from leaf nitrogen assimilation products was similar to that originating from root nitrogen assimilation products, indicating that the total organic nitrogen contribution to the stems from leaves and roots was similar.

[0136] According to f in Table 8 leafstem and 1–f leafstem Combining the biomass and nitrogen content of the roots, stems and leaves of mulberry seedlings before and after the experimental treatment, the total nitrogen assimilation of the leaves and roots of mulberry seedlings during the entire experimental treatment period can be calculated, see Table 9.

[0137] Table 9 Total nitrogen assimilation in leaves and roots of mulberry seedlings under nitrate nitrogen treatment

[0138]

[0139] According to the total nitrogen assimilation of leaves and roots of mulberry seedlings at different nitrate nitrogen concentrations in Table 9, the contribution of nitrate assimilation of leaves and roots of mulberry seedlings at different nitrate nitrogen concentrations can be calculated, see Table 10.

[0140] Table 10 Nitrate assimilation contribution of leaves and roots of whole mulberry seedlings under nitrate nitrogen treatment

[0141]

[0142] As shown in Table 10, when nitrate nitrogen concentrations ranged from 0.5 to 8 mM, the contribution of nitrate assimilation in the leaves of mulberry seedlings was greater than that in the roots, indicating that nitrate assimilation in mulberry seedlings primarily occurs in the leaves. Therefore, using stable nitrogen isotope technology, we can quantify the contribution of nitrate assimilation in the leaves and roots of mulberry seedlings at different nitrate nitrogen concentrations, providing a deeper understanding of nitrogen metabolism in mulberry seedlings.

[0143] In summary, the stable nitrogen isotope fractionation of nitrate assimilation products throughout woody plants can be used to assess the inorganic nitrogen supply and demand relationship of woody plants under different nitrate nitrogen concentrations. Comparing two examples, we found that the nitrate nitrogen concentrations at which inorganic nitrogen supply and demand balance was approached in paper mulberry and mulberry seedlings differed between 0.5 and 8 mM nitrate nitrogen concentrations. The inorganic nitrogen supply of paper mulberry seedlings approached its demand at 8 mM nitrate nitrogen concentration, while the inorganic nitrogen supply of mulberry seedlings approached its demand at 2 mM nitrate nitrogen concentration. Overall, the nitrate nitrogen demand of paper mulberry seedlings was greater than that of mulberry seedlings. Therefore, assessing the inorganic nitrogen supply and demand relationship under different nitrate nitrogen concentrations based on the stable nitrogen isotope fractionation of nitrate nitrogen assimilation products throughout woody plants provides a theoretical basis for the scientific management of nitrate nitrogen supply in woody plants.

[0144] The total amount of nitrogen assimilated by leaves and roots of woody plants can be estimated using the isotope mass balance equation and the two-terminal isotope mixing model. This allows for the assessment of the contribution of leaves and roots to nitrate assimilation in woody plants when nitrate nitrogen is the sole nitrogen source. Comparing two case studies, we found that nitrate assimilation in mulberry seedlings primarily occurred in leaves when nitrate nitrogen concentrations ranged from 0.5 to 8 mM. In contrast, the primary site of nitrate assimilation in paper mulberry seedlings depended on the external nitrate concentration. At low nitrate nitrogen concentrations, nitrate assimilation in paper mulberry seedlings primarily occurred in the roots; at high nitrate concentrations, nitrate assimilation in paper mulberry seedlings primarily occurred in the leaves. Paper mulberry seedlings adjust their nitrogen metabolism based on the external nitrate supply. In particular, in low nitrogen environments, paper mulberry seedlings accelerate nitrogen assimilation in their roots, facilitating root growth and laying the morphological foundation for their adaptation to karst environments. Furthermore, quantifying the contribution of leaves and roots to nitrate assimilation in woody plants under varying nitrate nitrogen concentrations provides a deeper understanding of nitrogen metabolism in woody plants.

[0145] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for evaluating the relationship between nitrate supply and demand and the distribution of nitrate assimilation products in woody plants, characterized in that: The following steps are involved: First, woody plant seedlings with consistent growth were selected for the experiment. Before the experiment, the dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of the woody plant seedlings were measured. The initial dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves of the woody plant seedlings were recorded as DW, l0 、N l0 and δ 15 N l0 , the initial dry weight, nitrogen content and stable nitrogen isotope value of the stem were recorded as DW s0 、N s0 and δ 15 N s0 The initial dry weight, nitrogen content and stable nitrogen isotope value of the root were recorded as DW r0 、N r0 and δ 15 N r0 , using DW l0 、N l0 , δ 15 N l0 、DW s0 、N s0 , δ 15 N s0 、DW r0 、N r0 and δ 15 N r0 , calculate the initial stable nitrogen isotope value and total nitrogen accumulation of the whole woody plant, which are recorded as δ 15 N whole-plant0 , and m whole-plant0 ; Second, the woody plant seedlings were planted in a nutrient solution containing only nitrate nitrogen. After a period of cultivation, the dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves, stems, and roots of the woody plant seedlings were measured. The dry weight, nitrogen content, and stable nitrogen isotope values ​​of the leaves of the woody plant seedlings after the experimental treatment were recorded as DW, l1 、N l1 and δ 15 N l1 , the stem dry weight, nitrogen content and stable nitrogen isotope value were recorded as DW s1 、N s1 and δ 15 N s1 , the dry weight, nitrogen content and stable nitrogen isotope value of the root were recorded as DW r1 、N r1 and δ 15 N r1 , using DW l1 、N l1 , δ 15 N l1 、DW s1 、N s1 , δ 15 N s1 、DW r1 、N r1 and δ 15 N r1 , calculate the stable nitrogen isotope value and total nitrogen accumulation of the whole woody plant after the experimental treatment, and record them as δ 15 N whole-plant1 , and m whole-plant1 ; Third, according to the isotope mass balance equation, based on δ 15 N whole-plant0 , δ 15 N whole-plant1 、m whole-plant0 and m whole-plant1 , calculate the stable isotope value of nitrogen assimilation products of the whole woody plant, recorded as δ 15 N assimilates ; Fourth, according to δ 15 N assimilates Calculate the stable nitrogen isotope fractionation value of the nitrogen assimilation products of the whole woody plant, denoted as Δ 15 N assimilates , and according to Δ 15 N assimilates The size of the nitrate supply and demand relationship of woody plants is assessed; Fifth, according to the isotope mass balance equation, using DW l0 、N l0 , δ 15 N l0 、DW l1 、N l1 and δ 15 N l1 , calculate the stable nitrogen isotope value of the leaf nitrogen assimilation product, recorded as δ 15 N leaf-assimilates , using DW s0 、N s0 , δ 15 N s0 、DW s1 、N s1 and δ 15 N s1 , calculate the stable nitrogen isotope value of the stem nitrogen assimilation product, denoted as δ 15 N stem-assimilates , using DW r0 、N r0 , δ 15 N r0 、DW r1 、N r1 and δ 15 N s1 , calculate the stable nitrogen isotope value of the nitrogen assimilation product of the root, recorded as δ 15 N root-assimilates ; Sixth, according to the two-terminal isotope mixing model, using δ 15 N leaf-assimilates , δ 15 N stem-assimilates and δ 15 N root-assimilates Calculate the proportion of new stem nitrogen in woody plants that comes from leaf nitrogen assimilation, denoted as f leafstem ; The part derived from nitrogen assimilation in roots is denoted as 1–f leafstem ; Seventh, according to f leafstem or 1–f leafstem ; Calculate the total amount of nitrogen assimilation products in the leaves and roots of woody plants, respectively, and record them as m leaf and m root , and then evaluate the contribution of leaves and roots to nitrate assimilation; Eighth, according to m leaf and m root , calculate the nitrate assimilation contribution of woody plant leaves, recorded as P, and the nitrate assimilation contribution of woody plant roots is 1–P.

2. The method for evaluating the nitrate supply and demand relationship and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the first step, δ 15 N whole-plant0 The calculation equation is as follows: δ 15 N whole-plant0 (‰)=(DW l0 ×N l0 ×δ 15 N l0 +DW s0 ×N s0 ×δ 15 N s0 +DW r0 ×N r0 ×δ 15 N r0 ) / (DW l0 ×N l0 +DW s0 ×N s0 +DW r0 ×N r0 ) m whole-plant0 The calculation equation is as follows: m whole-plant0 =DW l0 ×N l0 +DW s0 ×N s0 +DW r0 ×N r0 。 3. The method for evaluating the nitrate supply and demand relationship and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the second step, the nutrient solution is prepared with nitrate as the only nitrogen source to an appropriate nitrogen concentration, and the stable nitrogen isotope value in the nitrate is greater than 20‰.

4. The method for evaluating the relationship between nitrate supply and demand and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the second step, δ 15 N whole-plant1 The calculation equation is as follows: δ 15 N whole-plant1 (‰)=(DW l1 ×N l1 ×δ 15 N l1 +DW s1 ×N s1 ×δ 15 N s1 +DW r1 ×N r1 ×δ 15 N r1 ) / (DW l1 ×N l1 +DW s1 ×N s1 +DW r1 ×N r1 ); m whole-plant1 The calculation equation is as follows: m whole-plant1 =DW l1 ×N l1 +DW s1 ×N s1 +DW r1 ×N r1 。 5. The method for evaluating the relationship between nitrate supply and demand and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the third step, δ 15 N assimilates The calculation equation is as follows: δ 15 N assimilates (‰)=(m whole-plant1 ×δ 15 N whole-plant1 -m whole-plant0 ×δ 15 N whole-plant0 ) / (m whole-plant1 -m whole-plant0 )。 6. The method for evaluating the relationship between nitrate supply and demand and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the fourth step, Δ 15 N assimilates The calculation equation is as follows: D 15 N assimilates (‰)=δ 15 N substrate -d 15 N assimilates Among them, δ 15 N substrate It is the stable nitrogen isotope value of nitrate nitrogen in chemical drugs.

7. The method for evaluating the relationship between nitrate supply and demand and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the fifth step, δ 15 N leaf-assimilates The calculation equation is as follows: δ 15 N leaf-assimilates (‰)=(DW l1 ×N l1 ×δ 15 N l1 -DW l0 ×N l0 ×δ 15 N l0 ) / (DW l1 ×N l1 -DW l0 ×N l0 ) δ 15 N stem-assimilates The calculation equation is as follows: δ 15 N stem-assimilates (‰)=(DW s1 ×N s1 ×δ 15 N s1 -DW s0 ×N s0 ×δ 15 N s0 ) / (DW s1 ×N s1 -DW s0 ×N s0 ) δ 15 N root-assimilates The calculation equation is as follows: δ 15 N root-assimilates (‰)=(DW r1 ×N r1 ×δ 15 N r1 -DW r0 ×N r0 ×δ 15 N r0 ) / (DW r1 ×N r1 -DW r0 ×N r0 )。 8. The method for evaluating the relationship between nitrate supply and demand and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the sixth step, f leaf stem The calculation equation is as follows:

9. The method for evaluating the relationship between nitrate supply and demand and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the seventh step, m leaf The calculation equation is as follows: m leaf =(DW l1 ×N l1 -DW l0 ×N l0 )+f leaf stem ×(DW s1 ×N s1 -DW s0 ×N s0 ) m root The calculation equation is as follows: m root =(DW r1 ×N r1 -DW r0 ×N r0 )+(1-f leaf stem )×(DW s1 ×N s1 -DW s0 ×N s0 )。 10. The method for evaluating the nitrate supply and demand relationship and the distribution of nitrate assimilation products in woody plants according to claim 1, characterized in that: In the eighth step, the calculation equation of P is as follows:

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