Evaluation of the effect of L-leucine in Plant growth promoters

As an essential branched-chain amino acid (BCAA) for humans, the functions of L-leucine in plant physiological metabolism have been gradually revealed in recent years. Acting as a plant growth promoter, it exhibits significant regulatory effects on plant growth and development, yield formation, and quality improvement by participating in nitrogen metabolism, regulating hormone balance, strengthening stress resistance defense systems, and optimizing nutrient absorption efficiency. This article systematically elaborates on the efficacy evaluation system of L-leucine as a plant growth promoter, including evaluation indicators, testing methods, action mechanisms, and application scenario optimization. Combined with indoor pot experiment and field verification data, it quantifies the application effects on different crops, providing a scientific basis for the research, development, and application of novel plant growth regulators in green agriculture.

I. Core Indicator System for Efficacy Evaluation

1. Growth and Development Indicators (Basic Evaluation Dimension)

Focusing on key stages of plant vegetative and reproductive growth, the impact of L-leucine on plant morphogenesis is quantified:

Vegetative Growth Indicators: Plant height, stem diameter (diameter at 10 cm above the base), leaf area index (LAI), root-shoot ratio, and total biomass (fresh weight/dry weight). Measured using a ruler, vernier caliper, and leaf area meter, data are continuously monitored at key growth stages such as seedling stage, jointing/elongation stage, and flowering stage to evaluate the promotion of plant nutrient accumulation.

Reproductive Growth Indicators: Days to flowering advancement, number of flowers per plant, fruit set rate, seed setting rate, single fruit weight/1000-grain weight, and total yield. For different crop types (grain crops: wheat, corn; cash crops: tomato, cucumber; fruit trees: strawberry, apple), the optimization effects on yield components are emphasized.

2. Physiological and Metabolic Indicators (Mechanism-Associated Dimension)

The action mechanism of L-leucine is revealed by determining key physiological and biochemical parameters in plants:

Nitrogen Metabolism-Related Indicators: Leaf chlorophyll content (SPAD value), nitrate reductase (NR) activity, glutamine synthetase (GS) activity, and soluble protein content. Chlorophyll content is measured in situ using a SPAD meter, and enzyme activities are detected by colorimetry, reflecting the enhancement of plant nitrogen absorption and assimilation efficiency by L-leucine.

Antioxidant Defense Indicators: Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as malondialdehyde (MDA) content. Determined by spectrophotometry, these indicators evaluate the effect of L-leucine on enhancing plant stress resistance (drought, salt stress, diseases).

Hormone Balance Indicators: Contents of indole-3-acetic acid (IAA), gibberellic acid (GA₃), cytokinin (CTK), and abscisic acid (ABA). Measured by high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA), these data analyze the regulatory effect of L-leucine on plant hormone synthesis and signal transduction.

3. Quality and Stress Resistance Indicators (Application Value Dimension)

Quality Indicators:

Grain crops: Protein content, starch content, and amino acid composition (determined by HPLC).

Fruit and vegetable crops: Vitamin C content (titration method), soluble sugar content (anthrone colorimetry), organic acid content (acid-base titration), and soluble solid content (refractometer method).

Cash crops (e.g., cotton): Fiber length, breaking strength, and micronaire value.

Stress Resistance Indicators:

Abiotic stress (drought/salt stress): Relative water content (RWC), electrolyte leakage (EL), and proline content under stress.

Biotic stress (diseases): Diseased plant rate, disease index, and activities of defense enzymes (polyphenol oxidase PPO, phenylalanine ammonia-lyase PAL).

II. Testing Methods and Experimental Design for Efficacy Evaluation

1. Experimental Design Principles

Test Materials: Representative crop varieties are selected (e.g., wheat variety Jimai 44, tomato variety Zhongza 105, corn variety Zhengdan 958). Seeds are sown after disinfection and germination to ensure consistency of test materials.

Treatment Gradients: L-leucine concentration gradients (0 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L, 800 mg/L) are set, with clear water as the blank control and commercial amino acid fertilizer as the positive control in some experiments.

Application Methods: Include foliar spraying (1~3 applications at key growth stages), soil base application (mixed with soil before sowing), seed soaking (soaking for 6~12 hours), and drip irrigation application (3~5 split applications during the growth period) to clarify the effect differences of different application methods.

Experimental Types:

Indoor pot experiments: Environmental conditions such as light (16h light/8h dark), temperature (25±2℃), and humidity (60%~70%) are controlled to focus on single-factor effects, facilitating mechanism analysis.

Field plot experiments: Representative soil types (loam, sandy loam) are selected, with 3 replications, plot area of 15~20 m², and randomized block design. The actual application effects under natural conditions are recorded for yield and quality verification.

2. Data Collection and Statistical Analysis

Sampling Time: Samples are collected every 7~10 days during the vegetative growth period and at key nodes of the reproductive growth period (flowering stage, fruit setting stage, maturity stage) to determine physiological indicators; yield and quality indicators are measured at maturity.

Sample Processing: Leaves, roots, fruits, and other samples are quickly frozen in liquid nitrogen and stored at -80℃ for the detection of enzyme activities, hormones, nutrients, and other indicators.

Statistical Methods: SPSS or R software is used for one-way analysis of variance (ANOVA), and multiple comparisons are performed using Duncan’s new multiple range test, with P＜0.05 considered statistically significant. Correlation analysis and principal component analysis (PCA) are used to screen key factors affecting efficacy.

III. Plant Growth Promotion Mechanisms of L-Leucine

1. Optimizing Nitrogen Metabolism and Improving Nutrient Use Efficiency

As a nitrogen source precursor, L-leucine can directly participate in plant protein synthesis and strengthen nitrogen absorption and assimilation by regulating the activity of key nitrogen metabolism enzymes:

Promotes root absorption of nitrate nitrogen (NO₃⁻) and ammonium nitrogen (NH₄⁺), upregulates the expression of root nitrate reductase (NR) and glutamine synthetase (GS), accelerates the conversion of nitrogen into amino acids and proteins, and reduces nitrogen loss.

As a branched-chain amino acid, it can synergize with other amino acids to optimize the amino acid composition in plants, improve protein synthesis efficiency, and promote plant dry matter accumulation. Pot experiments show that foliar spraying of 100 mg/L L-leucine on wheat increases the soluble protein content of seedling leaves by 28% and the chlorophyll SPAD value by 15%.

2. Regulating Hormone Balance and Promoting Growth and Development

L-leucine achieves precise regulation of growth and development by regulating the synthesis and signal transduction of plant hormones:

Promotes the synthesis of indole-3-acetic acid (IAA) and gibberellic acid (GA₃), upregulates cytokinin (CTK) content, and inhibits the accumulation of abscisic acid (ABA), accelerating cell division and elongation, promoting plant height growth, stem diameter thickening, and flower bud differentiation.

During the reproductive growth stage, it can increase the IAA/ABA ratio at the fruit setting stage, reduce flower and fruit drop, and improve fruit set rate and seed setting rate. Tomato field experiments show that drip irrigation with 200 mg/L L-leucine increases the fruit set rate by 18% and single fruit weight by 22%.

3. Strengthening Antioxidant Defense and Enhancing Stress Resistance

L-leucine reduces cell damage caused by stress by activating the plant antioxidant system:

Acts directly as an antioxidant to scavenge reactive oxygen species (ROS) or upregulates the activity of antioxidant enzymes such as SOD, POD, and CAT, reducing MDA content, minimizing cell membrane lipid peroxidation, and maintaining cell structure integrity.

Promotes the synthesis of osmotic adjustment substances such as proline, improves the osmotic adjustment capacity of plants under drought and salt stress, and reduces water loss and ionic toxicity. Under salt stress (150 mmol/L NaCl), corn seeds soaked in 150 mg/L L-leucine show a 30% increase in relative water content and a 25% decrease in electrolyte leakage rate.

4. Improving Soil Microenvironment and Promoting Root Growth

The application of L-leucine can affect the soil microecology through root exudates:

Promotes the proliferation of beneficial soil microorganisms (e.g., nitrogen-fixing bacteria, phosphorus-solubilizing bacteria), improving the availability of soil nutrients such as nitrogen, phosphorus, and potassium.

Stimulates root elongation and lateral root germination, increases root surface area, and enhances root absorption capacity, forming a virtuous cycle of "root growth-nutrient absorption-plant robustness." In cucumber pot experiments, soil base application of 200 mg/L L-leucine increases root fresh weight by 45% and root-shoot ratio by 32%.

IV. Application Effect Verification on Different Crops

1. Grain Crops (Wheat, Corn)

Wheat: Foliar spraying of 100~200 mg/L L-leucine (jointing stage + booting stage) increases plant height by 8%~12%, 1000-grain weight by 10%~15%, and total yield by 12%~18%. Meanwhile, the grain protein content increases by 2%~3%, starch content slightly increases, and quality is significantly improved.

Corn: Seed soaking (8 hours) + jointing stage drip irrigation with 150~300 mg/L L-leucine increases stem diameter by 10%~14%, leaf area index by 15%~20%, reduces bald tip rate by 20%~30%, and increases total yield by 15%~22%. Under salt stress, the stress resistance effect is particularly significant, reducing yield loss by more than 30%.

2. Fruit and Vegetable Crops (Tomato, Cucumber, Strawberry)

Tomato: Drip irrigation with 200 mg/L L-leucine (seedling stage + flowering stage + fruit setting stage) increases fruit set rate by 15%~20%, number of fruits per plant by 12%~16%, and total yield by 18%~25%. The fruit vitamin C content increases by 20%~28%, soluble sugar content by 10%~15%, organic acid content decreases by 8%~12%, and taste is improved.

Cucumber: Foliar spraying of 150 mg/L L-leucine (vining stage + early fruiting stage) increases the number of fruits per plant by 10%~14%, single fruit weight by 8%~12%, and total yield by 13%~18%. The downy mildew disease index decreases by 25%~30%, and disease resistance is significantly enhanced.

Strawberry: Foliar spraying of 100 mg/L L-leucine (bud stage + fruit expansion stage) advances fruit maturity by 3~5 days, increases single fruit weight by 12%~18%, soluble solid content by 15%~20%, and reduces malformed fruit rate by 20%~25%.

3. Cash Crops (Cotton, Rapeseed)

Cotton: Foliar spraying of 200~300 mg/L L-leucine (bud stage + boll stage) increases the number of bolls per plant by 8%~12%, boll weight by 10%~15%, fiber length by 3%~5%, breaking strength by 4%~6%, and optimizes micronaire value to the suitable range of 2.7~3.2.

Rapeseed: Seed soaking + seedling stage foliar spraying with 150 mg/L L-leucine increases plant height by 7%~10%, number of pods per plant by 12%~16%, number of seeds per pod by 8%~12%, 1000-grain weight by 5%~8%, total yield by 10%~15%, and oil content by 2%~3%.

V. Application Optimization and Precautions

1. Application Technology Optimization

Concentration Adaptation: The suitable application concentration for most crops is 100~300 mg/L. Excessively high concentrations (>500 mg/L) may cause excessive plant growth and reduced stress resistance, requiring adjustment according to crop type and growth stage.

Application Timing: Application during the vegetative growth period (seedling stage, jointing stage) focuses on promoting plant growth, while application during the reproductive growth period (flowering stage, fruit setting stage/booting stage) focuses on improving yield and quality. Combined application at key growth stages achieves better results.

Application Methods: Foliar spraying is convenient and effective, suitable for large-scale promotion; soil base application and drip irrigation are more conducive to root absorption, suitable for protected agriculture and arid areas; seed soaking can improve germination rate and seedling stress resistance, being a low-cost and high-benefit application method.

2. Compound Synergy Strategies

Compound with Macronutrient Fertilizers (NPK): L-leucine can improve nutrient absorption efficiency, reducing chemical fertilizer usage by 10%~20%.

Compound with Micronutrients (Zinc, Iron, Boron): Promotes the chelation and absorption of micronutrients, alleviating nutrient deficiency symptoms.

Compound with Biofertilizers: Synergistically improves the soil microenvironment, enhances the activity of beneficial microorganisms, and strengthens stress resistance and growth promotion effects.

Avoid Mixing with Strongly Alkaline Pesticides/Fertilizers (pH＞8.5): To prevent L-leucine degradation and reduced efficacy.

3. Environmental Adaptability Precautions

L-leucine exhibits optimal stability and significant efficacy when soil pH is in the range of 5.5~7.5. For acidic soils (pH＜5.5), lime can be used to adjust pH before application.

Under high-temperature and strong-light conditions (temperature＞35℃), foliar spraying should be carried out in the early morning or evening to avoid component decomposition due to direct strong light. Meanwhile, 0.1%~0.2% adjuvant (e.g., Tween 80) can be added to improve leaf adsorption.

Under drought conditions, application should be combined with irrigation or drip irrigation to ensure effective absorption of L-leucine by plants.

VI. Challenges and Future Development Directions

1. Existing Challenges

Insufficient In-Depth Mechanism Analysis: The molecular mechanisms by which L-leucine regulates plant hormone synthesis and stress resistance signaling pathways have not been fully clarified, requiring further verification through transcriptomics and metabolomics.

Complex Influences of Environmental Factors: The effects of soil type, climate conditions, and crop varieties on efficacy vary, lacking universal application standards.

Needs for Formulation Technology Improvement: Most existing products are single amino acid formulations with limited stability and duration of action. There is a need to develop slow-release and controlled-release formulations to extend the action time.

2. Future Development Directions

Deepening Molecular Mechanism Research: Using gene editing, proteomics, and other technologies to identify key genes and regulatory networks of L-leucine action, providing theoretical support for precise application.

Developing Compound Formulations: Developing composite formulations of L-leucine with other branched-chain amino acids (isoleucine, valine), plant extracts, and biostimulants to achieve synergistic effects.

Intelligent Application Technology: Combining crop nutrition diagnosis and soil testing data, optimizing application concentration, timing, and methods through big data and artificial intelligence to achieve personalized and precise regulation.

Green and Sustainable Applications: Promoting the application of L-leucine in organic agriculture and ecological agriculture, reducing dependence on chemical pesticides and fertilizers, and supporting green agricultural development.

As a novel plant growth promoter, L-leucine exhibits significant effects in promoting growth, increasing yield, and optimizing quality of grain, fruit and vegetable, and cash crops by optimizing nitrogen metabolism, regulating hormone balance, strengthening stress resistance defense, and improving soil microenvironment. It also has advantages such as environmental friendliness, high biocompatibility, and low application cost. Through scientific experimental design and efficacy evaluation, clarifying the suitable application concentration, timing, and method for different crops, and combining compound synergy strategies, its application value can be further improved. In the future, with in-depth mechanism analysis and innovative formulation technologies, L-leucine will have broader application prospects in green agriculture, providing important technical support for ensuring food security and sustainable agricultural development.