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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Effect of Exogenous Hormones on the Growth of Alfalfa Seedlings under Phosphate Starvation Stress

N
Na Guo1
J
Jiarong Li1
M
Mingjiu Wang1,2
X
Xiaowei Huo1
L
Lin Bian1
Z
Zhenyi Li1,*
Z
Zhiqiang Zhang1,*
1College of Grassland Science, Inner Mongolia Agricultural University, Technology Engineering Center of Drought and Cold-Resistant Grass Breeding in North of the National Forestry and Grassland Administration, Key Laboratory of Grassland Resources of the Ministry of Education, Hohhot 010010, China.
2National Center of Pratacultural Technology Innovation (under preparation), Hohhot 010010, China.

  • Submitted03-07-2025|

  • Accepted15-08-2025|

  • First Online 20-09-2025|

  • doi 10.18805/LRF-885

ABSTRACT

Background: Alfalfa (Medicago sativa) is a high-quality legume forage. Phosphorus (P) is essential for plant productivity. Approximately half of global arable soils exhibit available-P deficiency, constraining plants growth and yield. This study aimed to explore how hormones mediate the responses of alfalfa to phosphate starvation.

Methods: We studied the morphological and physiological changes alfalfa (Zhongmu No.3) seedlings treated with auxin, ethylene and gibberellin under conditions of phosphate deficiency. 20-day-old alfalfa seedlings were exposed to varying concentrations of hormones in Hoagland solution. Changes in root morphology, photosynthesis, physiological characters and the levels of phosphate starvation induced (PSI) genes expression were monitored.

Result: IAA stimulated lateral-root growth (1.16-fold vs. control), up-regulated the organic phosphate transporter (PHT1-4) and purple acid phosphatase (PAP23) and inhibited primary-root elongation. Ethylene promoted the growth of root hairs and 10 μM aminocyclopropane-1-carboxylic acid (ACC) increased the elongation of roots and their surface area.1 μM ACC boosted shoot and root acid phosphatase activity and up-regulated 1-aminocyclopropane-1-carboxylate oxidase (ACO), whereas 5 μM gibberellin A3 (GA3) promoted stem and leaf growth, increasing stem diameter by 16.10% and leaf area by 36.90%, while elevating sulfoquinovosyl diacylglycerol (SQD2) content and DELLA expression. There was a strong negative correlation (-0.79) between the plant height and root-to-shoot ratio and a positive correlation (0.62) between the number of lateral roots and the activity of acid phosphatase (ACP) in the roots. The most effective concentrations of hormones was 1 μM ACC, which enhanced the plant height, stem diameter, lateral root formation, chlorophyll biosynthesis and nitrogen content and induced the levels of expression of PSI and key genes in hormone signaling.

INTRODUCTION

Phosphorus (P) is a necessary macronutrient for plants and it is involved in processes, such as photosynthesis and enzyme regulation (Sulieman et al., 2013; Prakash et al., 2025; Journals et al., 2024). However, nearly half of the global arable land is deficient in available phosphate (Hynst et al., 2024; Yang et al., 2024). This widespread deficiency in available phosphate significantly hinders plant growth, development and yield due to stunted development, including reduced height and leaf area (Wang et al., 2023; Puga et al., 2024; Nussaume et al., 2024; Balboa et al., 2024). The root system is essential for nutrient and water uptake and root architecture strongly governs inorganic phosphate (Pi) acquisition (Comte et al., 2013; Ren et al., 2023). Plants absorb Pi through their roots and respond to Pi deficiency by remodeling root morphology and physiology (Kumar et al., 2019; Nahar et al., 2022; Chiou et al., 2011).
       
Exogenous auxin, ethylene and gibberellins collectively enhance plant growth and phosphorus-use efficiency (PUE) under low-phosphate (LP) stress by modulating root architecture, Pi transporters and the phosphate-starvation response (Xu et al., 2020; Han et al., 2022; Mimura et al., 2024; Anfang et al., 2021; Zhao et al., 2021).  Auxin adjusts the root architecture and distribution of dry matter under low available phosphate stress, which supports the development of lateral roots (Nacry et al., 2005; Jain et al., 2007; Xia et al., Wonget_al2023). Auxin reallocates dry matter and stimulates lateral-root formation, mitigating P deficiency (Di et al., 2018). Under LP stress, it triggers ethylene production and citric-acid exudation in Medicago truncatula roots, enhancing Pi uptake (Chen et al., 2023). In Cunninghamia lanceolata seedlings, ethylene boosts root growth and Pi absorption. Through its receptor ethylene response sensor 1 (ERS1) and the transcription factor ethylene insensitive 3 (EIN3), ethylene up-regulates root-hair genes, promoting hair formation and improving phosphorus-acquisition efficiency (Zhang et al., 2021). Gibberellins enhance plant-specific photosynthetic and nitrogen-metabolizing enzyme activities, increasing plant height and leaf area (Fonouni-Farde  et al., 2019; Brini et al., 2022). Phosphate deficiency triggers complex crosstalk among gene expression, root architecture and hormonal signaling (Nasr Esfahani and Sonnewald, 2024). In gibberellin- treated Arabidopsis thaliana, overexpression of specific genes represses phosphorus-starvation-induced genes, modifies root morphology, reduces Pi uptake and acid phosphatase (ACP) activity and consequently lowers shoot phosphorus content (Gong et al., 2024; Jiang et al., 2007).
               
Alfalfa (Medicago sativa) requires 10-15 mg kg-1 of available phosphate (Pi) for optimal growth, yet soil Pi frequently falls below this threshold, resulting in chronic deficiency. The hormonal mechanisms underlying alfalfa’s response to LP remain unresolved. We therefore applied graded concentrations of auxin, ethylene and gibberellin to Pi-limited alfalfa to elucidate the associated physiological and morphological changes and to evaluate the potential of phytohormone supplementation for alleviating Pi deficiency.

MATERIALS AND METHODS

Plant material
 
This study was carried out at the Key Laboratory of Grassland Resources of the Ministry of Education, located at Inner Mongolia Agricultural University in Hohhot, China, from December 2023 to December 2024. Alfalfa Zhongmu No. 3 seeds were germinated for 8 days and then grown in Hoagland solution at pH 6.0 for 12 days (Xia et al., 2024).  Control groups received 1,000 μM KH2PO4 (normal Pi) or 10 μM KH2PO4 (LP). The treated groups were established under LP conditions with auxin (0.1, 1 and 10 μM IAA), ethylene (1, 10 and 50 μM ACC), or gibberellin (0.1, 5 and 10 μM GA3). To eliminate differences in K+ concentration between treatments, K+ in the LP treatment was provided by 1 mmol L-1 K2SO4 (Zhu et al., 2022). The plants were maintained in an artificial climate chamber at 22oC with a 16 h/8 h light/dark cycle. The Hoagland solution was refreshed every 3 days (Chen et al., 2017).
 
Measurement of the morphological characters
 
Height, stem diameter (3, 6, 12 d), leaf area (YaXin, Beijing, China) and the dry weight of the shoots and roots was determined. The roots were separated, imaged by an image scanner and analyzed for their length, surface area and numbers of lateral roots using WinRHIZO software (v.5.0, Regent Instruments, Quebec City, Canada). Root hairs from the seedlings were observed using a microscope (BX41, Olympus, Japan). Three biological repetitions that contained four technical repetitions per repetition, respectively, were established in this study (Ma et al., 2023).
 
Agronomic and physiological character measurements
 
The net photosynthetic rate (Pn) and transpiration rate (Tr) were measured at 3, 6, 12 d (LI-6400XT; LI-COR, Lincoln, NE, USA). The gas flow was set at 500 μmol·s-1 and the leaf chamber was illuminated by a 3 cm x 2 cm red and blue LED. Chlorophyll and nitrogen were quantified with a portable chlorophyll meter (n = 5). Leaf enzymes were assayed with p-nitrophenyl phosphate (pNPP) as substrate to generate p-nitrophenol, (PNP) on 405 nm; ACP activities in shoots and roots were quantified with an ACP Activity Assay Kit (ACP-1-W, Comin, Nanjing, China).
 
Analysis of the expression of the PSR genes
 
Total RNA was extracted from the shoots and roots using a FastPure Universal Plant Total RNA Isolation Kit (RC411-01, Vazyme Biotech Co., Ltd., Nanjing, China). quantified with NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), reverse transcription to complementary DNA (cDNA) using a HiScript II kit (Vazyme) and stored at -20oC. Gene-specific primers were designed with Primer Premier 5.0 and Primer-BLAST (NCBI) to ensure specificity. In addition, the primer pair of actin has been reported in previous  studies (Zhou et al., 2019). The amplification program was as follows: 95oC for 30 s, followed by 95oC for 5 s, 60oC for 30 s, and 95oC for 15 s, repeated for 40 cycles. Relative gene expression was calculated using the 2-ΔΔCt method (Table 1).

Table 1: Gene primers for the real-time quantitative reverse-transcription PCR.


 
Statistical analysis
 
The data was organized using Microsoft Excel 2020 (Redmond, WA, USA). Statistical analysis appears to have been performed with appropriate tools (SPSS, PCA,Origin) (IBM, Inc, Armonk, NY, USA and OriginLab, Northampton, MA, USA)
The affiliation function is as follows:
 
  
 
The formula for weighting is as follows:.
 
 
  
Finally, the combined evaluation value (d) is calculated using the following formula (d) value:
 
 
  
Where,
U(X) = Affiliation function value of an evaluation indicator.
X = denotes the measured value of that indicator.
       
The symbols Xmax and Xmin indicate the maximum and minimum values for each respective indicator. j = 1,2,…,n. Wj denotes the weighting formula and Pj denotes the contribution of the jth principal component.

RESULTS AND DISCUSSION

Influence of the hormones on agronomic traits
 
Pi deficiency differentially modulated hormone mediated growth of alfalfa seedlings at 3, 6 and 12 d, resulting in reduced height, leaf area, stem diameter and shoot biomass (Fig 1). IAA (0.1 and 1 μM) alleviated Pi-deficiency growth inhibition, elevating height, stem diameter and shoot biomass within 3-12 d. (Fig 1a, c, d). 0.1 μM IAA boosted height by 25% and 1 μM IAA increased stem diameter by 27% at 6 d and leaf area by 55% at 12 d relative to LP. 10 μM IAA suppressed leaf growth. ACC inhibited height except for 1 μM at 12 d versus control, whereas 1 and 10 μM ACC significantly increased height and stem diameter at 6 and 12 d. (Fig 1b, c). Treatment with 50 μM ACC inhibited the biomass but increased the root-to-shoot ratio (P<0.05) (Fig 1e). An increase in plant height would normally be expected with GA3 application. Instead, a reduction in leaf area and dry weight is observed with higher GA3 concentrations (Fig 1a). The stem diameter at 12 d after treatment with 5 and 10 μM GA3 and the aboveground weight from 6 d to 12 d treated with 0.1 and 5 μM GA3 were significantly enhanced (Fig 1c, d). At 6 d, 0.1 and 5 μM GA3  elevated shoot biomass above +Pi levels and 5 μM GA3  further thickened stems by 16.10% and expanded leaf area by 36.90% (Fig 1d).

Fig 1: Influence of plant hormones on the agronomic traits of alfalfa.


 
Influence of hormones on the morphological charact-eristics of the roots
 
Among the three hormones, auxin and ethylene enlarged the root system by increasing root surface area, lateral root number and total root length, with 1 μM IAA and 10 μM ACC providing the greatest enhancement. 1 µM ACC resulted in the most significant increase in dry weight of underground plant (Fig 2a). Treatment with 1 µM IAA increased alfalfa root surface area 1.25-fold, lateral root number 2.16-fold and total root length 0.36-fold under phosphorus deficiency, whereas 10 µM IAA suppressed root weight, surface area and total root length (Fig 2b, d). Treatment with 1 and 10 μM ACC promoted the growth of root hairs and lateral roots, while 50 μM ACC inhibited the growth of lateral roots (Fig 2c, e).10 µM ACC increased root surface area by 28% and maximized cumulative root elongation. Among GA3 treatments, 0.1 µM GA3 raised root dry weight to 1.29-fold of the +P control at day 3, whereas 5 µM GA3  significantly extended total root length without affecting lateral-root density. ACC promoted root growth in a concentration-dependent manner over 3, 6 and 12 days (Fig 2e).

Fig 2: Influence of exogenous hormones on the morphological characteristics of alfalfa roots.


 
Effects of hormones on the photosynthetic traits
 
IAA reduced the Pn of the alfalfa seedlings and 0.1 μM yielded the lowest Pn. It also significantly increased the Tr, which peaked on day 6 at 3.4-fold the normal levels of phosphorus. Conversely, 10 μM IAA suppressed the Tr (Fig 3a). The Pn peaked at 10 μM ACC but was hindered by 1 and 50 ìM ACC. The addition of 1 μM ACC significantly increased the early-stage Tr in phosphorus-stressed seedlings but later decreased. Treatment with 1 μM ACC increased the biosynthesis of chlorophyll by 12.80% and the uptake of nitrogen by 13.60%. Treatment with 0.1 μM GA3 resulted in the highest Pn, which was 1.82-fold that of the control, while 10 μM GA3 showed the lowest rate, which was 1.51-fold lower. Treatment with 0.1 μM GA3 decreased the Tr but increased the biosynthesis of chlorophyll by 21.30% and the uptake of nitrogen by 14.80% (Fig 3c,d).

Fig 3: Influence of exogenous hormones on the photosynthetic traits of alfalfa.


 
Influence of hormones on the activity of ACP
 
Hormones differentially modulated ACP activity: Early IAA or GA3  suppressed shoot ACP but enhanced root secretion. After 12 d, 10 µM IAA elevated shoot ACP to 91.74 (P<0.05). The roots treated with 1 μM ACC had 10.51 μmol/min/g ACP, which was 3.06-fold that of the control. 10 µM GA3 increased shoot ACP, whereas 5-10 µM GA3 reduced root ACP (Fig 4).

Fig 4: Influence of exogenous hormones on the physiological parameters of alfalfa.


 
Correlation analysis of the different characters and a membership function analysis
 
Under P deficiency, a Pearson correlation analysis showed that plant height (-0.79) and root-to-shoot ratio were inversely related; lateral-root number correlated positively with ACP (0.62), root length (0.93) and surface area (0.76). Chlorophyll and nitrogen contents were tightly linked (0.97) and root surface area associated with Pn (0.64), which suggested the development of a root system that affects the photosynthetic efficiency and overall growth (Fig 5).

Fig 5: Correlation analysis of the morphological and physiological parameters in alfalfa.


       
Membership-function analysis of nine morphological traits across 11 treatments (≥85% cumulative variance) extracted four principal components (≥89.45% contribution) to rank the optimal hormone concentrations for low-P-stressed alfalfa seedlings.1 μM ACC (D = 0.7303), 10 μM ACC (0.6258) and 5 μM GA3 (0.6004) were the top three hormone concentrations for promoting shoot growth under P deficiency. Six treatments (all ethylene, 0.1 and 5 μM GA3, 1 μM IAA) exceeded normal-P controls. Low-P ethylene, especially 1 μM ACC, surpassed normal growth. The concentrations of auxin promoted growth, whereas 10 μM GA3 inhibited it, yielding poorer performance than P-stressed plants (Table 2).

Table 2: Affiliation function values of the different treatments for the morphological traits in alfalfa.


 
Effects of hormones on the PSI genes and the levels of expression of the key hormone signaling genes
 
Roots expressed key PSI genes (PHT1-4, PAP23, SQD2). IAA (10 μM) progressively up-regulated PHT1-4 (P<0.05); 1 μM ACC down-regulated PHT1-4, whereas 5 μM GA3  up-regulated it. PAP23 was induced by LP stress compared to the conditions of normal phosphate. Seedlings treated with 10 μM IAA or ACC for 12 h significantly up-regulated PAP23. The highest PAP23 level was reached at 6 d with 5 μM GA3. SQD2 expression increased with auxin and ethylene concentrations, was lowest under 1 μM ACC and was significantly  higher with 5 μM GA3 than all other treatments (Fig 6).

Fig 6: The relative levels of expression of genes.


       
1 μM IAA optimally induced AUX expression. Peak AUX expression at 3 d under 0.1 or 1 μM IAA.ACO was also induced by ACC treatments from 12 h to 12 d. 1 μM ACC elicited maximal ACO expression at 3 d. DELLA was expressed at the highest levels in seedlings that appeared to have been exposed to 0.1 μM GA3. However, 5 μM GA3 induced the peak expression after 3 days (Table 3).

Table 3: Expression of different exogenous hormones on the genes related to the roots of alfalfa.


       
The supply of phosphate directly affects the growth and development of plants through the biosynthesis of nucleic acids, proteins, energy-rich phosphate and the enhancement of photosynthesis and others (Iqbal et al., 2023; Salim et al., 2023). Phosphate starvation stress led to the increase in the activity of ACP, the elongation of roots and the production of organic acids and phosphate transporters (Xu et al., 2022; Dokwal et al., 2022). Increased phosphate uptake and transport contribute to the maintenance of stable phosphorus levels within the plant, thereby facilitating an improved adaptation to phosphate deficiency (Soumya et al., 2022; Lian et al., 2023). In this study, the height, stem thickness and leaf area of alfalfa were significantly reduced in conditions of phosphorus deficiency compared to plants grown under normal levels. This is probably because the alfalfa plants prioritized the allocation of phosphorus by restricting the growth of their stems and leaves to maintain the normal development of the plants. Nevertheless, the application of phytohormones enhanced the allocation of nutrients among the stems, leaves and root systems (Du et al., 2024; Yan et al., 2024; Nambara, 2021).
       
Exogenous phytohormones modulate biomass partitioning and root–shoot allocation under LP stress (Groenewald et al., 2019; Shi et al., 2017; Lei et al., 2022). Whereas 5 μM GA3 markedly increased shoot height and stem diameter-effects counteracted by 50 μM ACC-low-phosphate stress alone elevated the alfalfa root-to-shoot ratio by 9.60%, highlighting adaptive resource reallocation. Phytohormones further remodel root architecture for improved P acquisition (Nussaume et al., 2024). GA3 inhibited shoot and lateral-root growth yet elongated primary roots, whereas 1 μM IAA and 10 μM ACC most effectively stimulated lateral-root proliferation and expanded root surface area, respectively. In LP-stressed plants, 10 μM ACC maximized transpiration and photosynthesis; 1 μM ACC and 0.1 μM GA3 restored chlorophyll and nitrogen contents; and 1 μM ACC elevated acid phosphatase activity and Pi uptake. Optimal shoot biomass accrued with 5 μM GA3, but the most robust root system-greater surface area, lateral-root number and total length-was achieved with 1 μM IAA and 10 μM ACC.
       
Phytohormones modulate gene expression and metabolic pathways, thereby shaping plant growth, development and responses to LP. The PHT1 gene, part of the phosphorus transport protein family, is crucial for the uptake, function and utilization of phosphorus. These effects promote the translocation of phosphorous to specific tissues. The PAPs are the key to the utilization of organic phosphorus in the soil and its redistribution within the plants. qRT-PCR quantification of PHT1-4, PAP23, SQD2, AUX, ACO and DELLA revealed that hormone treatments elevated Pi-transporter abundance and up-regulated PAP23 and SQD2 in alfalfa. Various hormone treatments modulated the expression of these genes, which affected the growth and adaptation to LP stress. The expression of AUX peaked with 1 μM IAA, while PHT1-4 and PAP23 peaked with 10 μM IAA. Treatment with 1 μM ACC upregulated ACO and 5 μM GA3 upregulated SQD2 and DELLA. SQD2 induction under   LP reallocates membrane lipids and improves P recycling. Transcription factors within hormone signaling cascades integrate these responses and optimized hormone doses enhance Pi acquisition, alleviating growth inhibition under P deficiency.

CONCLUSION

Under phosphate deficiency, IAA enhances lateral-root initiation and elongation while suppressing primary-root extension. ACC promotes both lateral-root emergence and root-hair elongation. GA3 stimulates primary-root elongation yet inhibits lateral-root formation. Hormonal priming with ACC, GA3 and IAA alleviated P stress in alfalfa. IAA up-regulated PHT1-4, PAP23 and AUX; ethylene induced ACO; and GA3 elevated SQD2 and DELLA. Lateral-root abundance correlated positively with root enzyme activity. Membership-function analysis identified 1 μM ACC as the most effective treatment, significantly enhancing seedling height, stem diameter, lateral-root number, chlorophyll content, nitrogen uptake and ACP activity, thereby boosting LP tolerance.

ACKNOWLEDGEMENT

We acknowledge the platform of Key Laboratory of Grassland Germplasm Innovation and Sustainable Utilization of Grassland Resources in Inner Mongolia, Key Laboratory of Forage Cultivation, Processing and High Efficient Utilization of Ministry of Agriculture,  China.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Funding
 
The work was supported by National Natural Science Foundation of China (32301484), Inner Mongolia Autonomous Region Natural Science Foundation General Program (2025MS03136), Grassland Talents Program of Inner Mongolia Autonomous Region, Scientific Research Foundation for Advanced Talents by Inner Mongolia  Agricultural University (NDYB2022-51), The First-Class Discipline Scientific Research Program of Inner Mongolia (IMAUCXQJ2023015), Capacity Building Project of the National Forestry and Grassland Engineering Technology Research Center for the Breeding of Drought- and Cold-Tolerant Grass Varieties in Northern China (BR221029, BR251016), 2023 National Center of Pratacultural Technology Innovation (under preparation) Major Innovation Platform Construction Project (CCPTZX2023B0701), National Key R and D Program-Intergovernmental International Science and Technology Innovation Cooperation: China-Mongolia Joint Research Project (2024YFE0113100) and Scientific Research Funding for Universities Directly under the Inner Mongolia Autonomous Region (BR22-12-07).

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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    Effect of Exogenous Hormones on the Growth of Alfalfa Seedlings under Phosphate Starvation Stress

    N
    Na Guo1
    J
    Jiarong Li1
    M
    Mingjiu Wang1,2
    X
    Xiaowei Huo1
    L
    Lin Bian1
    Z
    Zhenyi Li1,*
    Z
    Zhiqiang Zhang1,*
    1College of Grassland Science, Inner Mongolia Agricultural University, Technology Engineering Center of Drought and Cold-Resistant Grass Breeding in North of the National Forestry and Grassland Administration, Key Laboratory of Grassland Resources of the Ministry of Education, Hohhot 010010, China.
    2National Center of Pratacultural Technology Innovation (under preparation), Hohhot 010010, China.

    • Submitted03-07-2025|

    • Accepted15-08-2025|

    • First Online 20-09-2025|

    • doi 10.18805/LRF-885

    ABSTRACT

    Background: Alfalfa (Medicago sativa) is a high-quality legume forage. Phosphorus (P) is essential for plant productivity. Approximately half of global arable soils exhibit available-P deficiency, constraining plants growth and yield. This study aimed to explore how hormones mediate the responses of alfalfa to phosphate starvation.

    Methods: We studied the morphological and physiological changes alfalfa (Zhongmu No.3) seedlings treated with auxin, ethylene and gibberellin under conditions of phosphate deficiency. 20-day-old alfalfa seedlings were exposed to varying concentrations of hormones in Hoagland solution. Changes in root morphology, photosynthesis, physiological characters and the levels of phosphate starvation induced (PSI) genes expression were monitored.

    Result: IAA stimulated lateral-root growth (1.16-fold vs. control), up-regulated the organic phosphate transporter (PHT1-4) and purple acid phosphatase (PAP23) and inhibited primary-root elongation. Ethylene promoted the growth of root hairs and 10 μM aminocyclopropane-1-carboxylic acid (ACC) increased the elongation of roots and their surface area.1 μM ACC boosted shoot and root acid phosphatase activity and up-regulated 1-aminocyclopropane-1-carboxylate oxidase (ACO), whereas 5 μM gibberellin A3 (GA3) promoted stem and leaf growth, increasing stem diameter by 16.10% and leaf area by 36.90%, while elevating sulfoquinovosyl diacylglycerol (SQD2) content and DELLA expression. There was a strong negative correlation (-0.79) between the plant height and root-to-shoot ratio and a positive correlation (0.62) between the number of lateral roots and the activity of acid phosphatase (ACP) in the roots. The most effective concentrations of hormones was 1 μM ACC, which enhanced the plant height, stem diameter, lateral root formation, chlorophyll biosynthesis and nitrogen content and induced the levels of expression of PSI and key genes in hormone signaling.

    INTRODUCTION

    Phosphorus (P) is a necessary macronutrient for plants and it is involved in processes, such as photosynthesis and enzyme regulation (Sulieman et al., 2013; Prakash et al., 2025; Journals et al., 2024). However, nearly half of the global arable land is deficient in available phosphate (Hynst et al., 2024; Yang et al., 2024). This widespread deficiency in available phosphate significantly hinders plant growth, development and yield due to stunted development, including reduced height and leaf area (Wang et al., 2023; Puga et al., 2024; Nussaume et al., 2024; Balboa et al., 2024). The root system is essential for nutrient and water uptake and root architecture strongly governs inorganic phosphate (Pi) acquisition (Comte et al., 2013; Ren et al., 2023). Plants absorb Pi through their roots and respond to Pi deficiency by remodeling root morphology and physiology (Kumar et al., 2019; Nahar et al., 2022; Chiou et al., 2011).
           
    Exogenous auxin, ethylene and gibberellins collectively enhance plant growth and phosphorus-use efficiency (PUE) under low-phosphate (LP) stress by modulating root architecture, Pi transporters and the phosphate-starvation response (Xu et al., 2020; Han et al., 2022; Mimura et al., 2024; Anfang et al., 2021; Zhao et al., 2021).  Auxin adjusts the root architecture and distribution of dry matter under low available phosphate stress, which supports the development of lateral roots (Nacry et al., 2005; Jain et al., 2007; Xia et al., Wonget_al2023). Auxin reallocates dry matter and stimulates lateral-root formation, mitigating P deficiency (Di et al., 2018). Under LP stress, it triggers ethylene production and citric-acid exudation in Medicago truncatula roots, enhancing Pi uptake (Chen et al., 2023). In Cunninghamia lanceolata seedlings, ethylene boosts root growth and Pi absorption. Through its receptor ethylene response sensor 1 (ERS1) and the transcription factor ethylene insensitive 3 (EIN3), ethylene up-regulates root-hair genes, promoting hair formation and improving phosphorus-acquisition efficiency (Zhang et al., 2021). Gibberellins enhance plant-specific photosynthetic and nitrogen-metabolizing enzyme activities, increasing plant height and leaf area (Fonouni-Farde  et al., 2019; Brini et al., 2022). Phosphate deficiency triggers complex crosstalk among gene expression, root architecture and hormonal signaling (Nasr Esfahani and Sonnewald, 2024). In gibberellin- treated Arabidopsis thaliana, overexpression of specific genes represses phosphorus-starvation-induced genes, modifies root morphology, reduces Pi uptake and acid phosphatase (ACP) activity and consequently lowers shoot phosphorus content (Gong et al., 2024; Jiang et al., 2007).
                   
    Alfalfa (Medicago sativa) requires 10-15 mg kg-1 of available phosphate (Pi) for optimal growth, yet soil Pi frequently falls below this threshold, resulting in chronic deficiency. The hormonal mechanisms underlying alfalfa’s response to LP remain unresolved. We therefore applied graded concentrations of auxin, ethylene and gibberellin to Pi-limited alfalfa to elucidate the associated physiological and morphological changes and to evaluate the potential of phytohormone supplementation for alleviating Pi deficiency.

    MATERIALS AND METHODS

    Plant material
     
    This study was carried out at the Key Laboratory of Grassland Resources of the Ministry of Education, located at Inner Mongolia Agricultural University in Hohhot, China, from December 2023 to December 2024. Alfalfa Zhongmu No. 3 seeds were germinated for 8 days and then grown in Hoagland solution at pH 6.0 for 12 days (Xia et al., 2024).  Control groups received 1,000 μM KH2PO4 (normal Pi) or 10 μM KH2PO4 (LP). The treated groups were established under LP conditions with auxin (0.1, 1 and 10 μM IAA), ethylene (1, 10 and 50 μM ACC), or gibberellin (0.1, 5 and 10 μM GA3). To eliminate differences in K+ concentration between treatments, K+ in the LP treatment was provided by 1 mmol L-1 K2SO4 (Zhu et al., 2022). The plants were maintained in an artificial climate chamber at 22oC with a 16 h/8 h light/dark cycle. The Hoagland solution was refreshed every 3 days (Chen et al., 2017).
     
    Measurement of the morphological characters
     
    Height, stem diameter (3, 6, 12 d), leaf area (YaXin, Beijing, China) and the dry weight of the shoots and roots was determined. The roots were separated, imaged by an image scanner and analyzed for their length, surface area and numbers of lateral roots using WinRHIZO software (v.5.0, Regent Instruments, Quebec City, Canada). Root hairs from the seedlings were observed using a microscope (BX41, Olympus, Japan). Three biological repetitions that contained four technical repetitions per repetition, respectively, were established in this study (Ma et al., 2023).
     
    Agronomic and physiological character measurements
     
    The net photosynthetic rate (Pn) and transpiration rate (Tr) were measured at 3, 6, 12 d (LI-6400XT; LI-COR, Lincoln, NE, USA). The gas flow was set at 500 μmol·s-1 and the leaf chamber was illuminated by a 3 cm x 2 cm red and blue LED. Chlorophyll and nitrogen were quantified with a portable chlorophyll meter (n = 5). Leaf enzymes were assayed with p-nitrophenyl phosphate (pNPP) as substrate to generate p-nitrophenol, (PNP) on 405 nm; ACP activities in shoots and roots were quantified with an ACP Activity Assay Kit (ACP-1-W, Comin, Nanjing, China).
     
    Analysis of the expression of the PSR genes
     
    Total RNA was extracted from the shoots and roots using a FastPure Universal Plant Total RNA Isolation Kit (RC411-01, Vazyme Biotech Co., Ltd., Nanjing, China). quantified with NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), reverse transcription to complementary DNA (cDNA) using a HiScript II kit (Vazyme) and stored at -20oC. Gene-specific primers were designed with Primer Premier 5.0 and Primer-BLAST (NCBI) to ensure specificity. In addition, the primer pair of actin has been reported in previous  studies (Zhou et al., 2019). The amplification program was as follows: 95oC for 30 s, followed by 95oC for 5 s, 60oC for 30 s, and 95oC for 15 s, repeated for 40 cycles. Relative gene expression was calculated using the 2-ΔΔCt method (Table 1).

    Table 1: Gene primers for the real-time quantitative reverse-transcription PCR.


     
    Statistical analysis
     
    The data was organized using Microsoft Excel 2020 (Redmond, WA, USA). Statistical analysis appears to have been performed with appropriate tools (SPSS, PCA,Origin) (IBM, Inc, Armonk, NY, USA and OriginLab, Northampton, MA, USA)
    The affiliation function is as follows:
     
      
     
    The formula for weighting is as follows:.
     
     
      
    Finally, the combined evaluation value (d) is calculated using the following formula (d) value:
     
     
      
    Where,
    U(X) = Affiliation function value of an evaluation indicator.
    X = denotes the measured value of that indicator.
           
    The symbols Xmax and Xmin indicate the maximum and minimum values for each respective indicator. j = 1,2,…,n. Wj denotes the weighting formula and Pj denotes the contribution of the jth principal component.

    RESULTS AND DISCUSSION

    Influence of the hormones on agronomic traits
     
    Pi deficiency differentially modulated hormone mediated growth of alfalfa seedlings at 3, 6 and 12 d, resulting in reduced height, leaf area, stem diameter and shoot biomass (Fig 1). IAA (0.1 and 1 μM) alleviated Pi-deficiency growth inhibition, elevating height, stem diameter and shoot biomass within 3-12 d. (Fig 1a, c, d). 0.1 μM IAA boosted height by 25% and 1 μM IAA increased stem diameter by 27% at 6 d and leaf area by 55% at 12 d relative to LP. 10 μM IAA suppressed leaf growth. ACC inhibited height except for 1 μM at 12 d versus control, whereas 1 and 10 μM ACC significantly increased height and stem diameter at 6 and 12 d. (Fig 1b, c). Treatment with 50 μM ACC inhibited the biomass but increased the root-to-shoot ratio (P<0.05) (Fig 1e). An increase in plant height would normally be expected with GA3 application. Instead, a reduction in leaf area and dry weight is observed with higher GA3 concentrations (Fig 1a). The stem diameter at 12 d after treatment with 5 and 10 μM GA3 and the aboveground weight from 6 d to 12 d treated with 0.1 and 5 μM GA3 were significantly enhanced (Fig 1c, d). At 6 d, 0.1 and 5 μM GA3  elevated shoot biomass above +Pi levels and 5 μM GA3  further thickened stems by 16.10% and expanded leaf area by 36.90% (Fig 1d).

    Fig 1: Influence of plant hormones on the agronomic traits of alfalfa.


     
    Influence of hormones on the morphological charact-eristics of the roots
     
    Among the three hormones, auxin and ethylene enlarged the root system by increasing root surface area, lateral root number and total root length, with 1 μM IAA and 10 μM ACC providing the greatest enhancement. 1 µM ACC resulted in the most significant increase in dry weight of underground plant (Fig 2a). Treatment with 1 µM IAA increased alfalfa root surface area 1.25-fold, lateral root number 2.16-fold and total root length 0.36-fold under phosphorus deficiency, whereas 10 µM IAA suppressed root weight, surface area and total root length (Fig 2b, d). Treatment with 1 and 10 μM ACC promoted the growth of root hairs and lateral roots, while 50 μM ACC inhibited the growth of lateral roots (Fig 2c, e).10 µM ACC increased root surface area by 28% and maximized cumulative root elongation. Among GA3 treatments, 0.1 µM GA3 raised root dry weight to 1.29-fold of the +P control at day 3, whereas 5 µM GA3  significantly extended total root length without affecting lateral-root density. ACC promoted root growth in a concentration-dependent manner over 3, 6 and 12 days (Fig 2e).

    Fig 2: Influence of exogenous hormones on the morphological characteristics of alfalfa roots.


     
    Effects of hormones on the photosynthetic traits
     
    IAA reduced the Pn of the alfalfa seedlings and 0.1 μM yielded the lowest Pn. It also significantly increased the Tr, which peaked on day 6 at 3.4-fold the normal levels of phosphorus. Conversely, 10 μM IAA suppressed the Tr (Fig 3a). The Pn peaked at 10 μM ACC but was hindered by 1 and 50 ìM ACC. The addition of 1 μM ACC significantly increased the early-stage Tr in phosphorus-stressed seedlings but later decreased. Treatment with 1 μM ACC increased the biosynthesis of chlorophyll by 12.80% and the uptake of nitrogen by 13.60%. Treatment with 0.1 μM GA3 resulted in the highest Pn, which was 1.82-fold that of the control, while 10 μM GA3 showed the lowest rate, which was 1.51-fold lower. Treatment with 0.1 μM GA3 decreased the Tr but increased the biosynthesis of chlorophyll by 21.30% and the uptake of nitrogen by 14.80% (Fig 3c,d).

    Fig 3: Influence of exogenous hormones on the photosynthetic traits of alfalfa.


     
    Influence of hormones on the activity of ACP
     
    Hormones differentially modulated ACP activity: Early IAA or GA3  suppressed shoot ACP but enhanced root secretion. After 12 d, 10 µM IAA elevated shoot ACP to 91.74 (P<0.05). The roots treated with 1 μM ACC had 10.51 μmol/min/g ACP, which was 3.06-fold that of the control. 10 µM GA3 increased shoot ACP, whereas 5-10 µM GA3 reduced root ACP (Fig 4).

    Fig 4: Influence of exogenous hormones on the physiological parameters of alfalfa.


     
    Correlation analysis of the different characters and a membership function analysis
     
    Under P deficiency, a Pearson correlation analysis showed that plant height (-0.79) and root-to-shoot ratio were inversely related; lateral-root number correlated positively with ACP (0.62), root length (0.93) and surface area (0.76). Chlorophyll and nitrogen contents were tightly linked (0.97) and root surface area associated with Pn (0.64), which suggested the development of a root system that affects the photosynthetic efficiency and overall growth (Fig 5).

    Fig 5: Correlation analysis of the morphological and physiological parameters in alfalfa.


           
    Membership-function analysis of nine morphological traits across 11 treatments (≥85% cumulative variance) extracted four principal components (≥89.45% contribution) to rank the optimal hormone concentrations for low-P-stressed alfalfa seedlings.1 μM ACC (D = 0.7303), 10 μM ACC (0.6258) and 5 μM GA3 (0.6004) were the top three hormone concentrations for promoting shoot growth under P deficiency. Six treatments (all ethylene, 0.1 and 5 μM GA3, 1 μM IAA) exceeded normal-P controls. Low-P ethylene, especially 1 μM ACC, surpassed normal growth. The concentrations of auxin promoted growth, whereas 10 μM GA3 inhibited it, yielding poorer performance than P-stressed plants (Table 2).

    Table 2: Affiliation function values of the different treatments for the morphological traits in alfalfa.


     
    Effects of hormones on the PSI genes and the levels of expression of the key hormone signaling genes
     
    Roots expressed key PSI genes (PHT1-4, PAP23, SQD2). IAA (10 μM) progressively up-regulated PHT1-4 (P<0.05); 1 μM ACC down-regulated PHT1-4, whereas 5 μM GA3  up-regulated it. PAP23 was induced by LP stress compared to the conditions of normal phosphate. Seedlings treated with 10 μM IAA or ACC for 12 h significantly up-regulated PAP23. The highest PAP23 level was reached at 6 d with 5 μM GA3. SQD2 expression increased with auxin and ethylene concentrations, was lowest under 1 μM ACC and was significantly  higher with 5 μM GA3 than all other treatments (Fig 6).

    Fig 6: The relative levels of expression of genes.


           
    1 μM IAA optimally induced AUX expression. Peak AUX expression at 3 d under 0.1 or 1 μM IAA.ACO was also induced by ACC treatments from 12 h to 12 d. 1 μM ACC elicited maximal ACO expression at 3 d. DELLA was expressed at the highest levels in seedlings that appeared to have been exposed to 0.1 μM GA3. However, 5 μM GA3 induced the peak expression after 3 days (Table 3).

    Table 3: Expression of different exogenous hormones on the genes related to the roots of alfalfa.


           
    The supply of phosphate directly affects the growth and development of plants through the biosynthesis of nucleic acids, proteins, energy-rich phosphate and the enhancement of photosynthesis and others (Iqbal et al., 2023; Salim et al., 2023). Phosphate starvation stress led to the increase in the activity of ACP, the elongation of roots and the production of organic acids and phosphate transporters (Xu et al., 2022; Dokwal et al., 2022). Increased phosphate uptake and transport contribute to the maintenance of stable phosphorus levels within the plant, thereby facilitating an improved adaptation to phosphate deficiency (Soumya et al., 2022; Lian et al., 2023). In this study, the height, stem thickness and leaf area of alfalfa were significantly reduced in conditions of phosphorus deficiency compared to plants grown under normal levels. This is probably because the alfalfa plants prioritized the allocation of phosphorus by restricting the growth of their stems and leaves to maintain the normal development of the plants. Nevertheless, the application of phytohormones enhanced the allocation of nutrients among the stems, leaves and root systems (Du et al., 2024; Yan et al., 2024; Nambara, 2021).
           
    Exogenous phytohormones modulate biomass partitioning and root–shoot allocation under LP stress (Groenewald et al., 2019; Shi et al., 2017; Lei et al., 2022). Whereas 5 μM GA3 markedly increased shoot height and stem diameter-effects counteracted by 50 μM ACC-low-phosphate stress alone elevated the alfalfa root-to-shoot ratio by 9.60%, highlighting adaptive resource reallocation. Phytohormones further remodel root architecture for improved P acquisition (Nussaume et al., 2024). GA3 inhibited shoot and lateral-root growth yet elongated primary roots, whereas 1 μM IAA and 10 μM ACC most effectively stimulated lateral-root proliferation and expanded root surface area, respectively. In LP-stressed plants, 10 μM ACC maximized transpiration and photosynthesis; 1 μM ACC and 0.1 μM GA3 restored chlorophyll and nitrogen contents; and 1 μM ACC elevated acid phosphatase activity and Pi uptake. Optimal shoot biomass accrued with 5 μM GA3, but the most robust root system-greater surface area, lateral-root number and total length-was achieved with 1 μM IAA and 10 μM ACC.
           
    Phytohormones modulate gene expression and metabolic pathways, thereby shaping plant growth, development and responses to LP. The PHT1 gene, part of the phosphorus transport protein family, is crucial for the uptake, function and utilization of phosphorus. These effects promote the translocation of phosphorous to specific tissues. The PAPs are the key to the utilization of organic phosphorus in the soil and its redistribution within the plants. qRT-PCR quantification of PHT1-4, PAP23, SQD2, AUX, ACO and DELLA revealed that hormone treatments elevated Pi-transporter abundance and up-regulated PAP23 and SQD2 in alfalfa. Various hormone treatments modulated the expression of these genes, which affected the growth and adaptation to LP stress. The expression of AUX peaked with 1 μM IAA, while PHT1-4 and PAP23 peaked with 10 μM IAA. Treatment with 1 μM ACC upregulated ACO and 5 μM GA3 upregulated SQD2 and DELLA. SQD2 induction under   LP reallocates membrane lipids and improves P recycling. Transcription factors within hormone signaling cascades integrate these responses and optimized hormone doses enhance Pi acquisition, alleviating growth inhibition under P deficiency.

    CONCLUSION

    Under phosphate deficiency, IAA enhances lateral-root initiation and elongation while suppressing primary-root extension. ACC promotes both lateral-root emergence and root-hair elongation. GA3 stimulates primary-root elongation yet inhibits lateral-root formation. Hormonal priming with ACC, GA3 and IAA alleviated P stress in alfalfa. IAA up-regulated PHT1-4, PAP23 and AUX; ethylene induced ACO; and GA3 elevated SQD2 and DELLA. Lateral-root abundance correlated positively with root enzyme activity. Membership-function analysis identified 1 μM ACC as the most effective treatment, significantly enhancing seedling height, stem diameter, lateral-root number, chlorophyll content, nitrogen uptake and ACP activity, thereby boosting LP tolerance.

    ACKNOWLEDGEMENT

    We acknowledge the platform of Key Laboratory of Grassland Germplasm Innovation and Sustainable Utilization of Grassland Resources in Inner Mongolia, Key Laboratory of Forage Cultivation, Processing and High Efficient Utilization of Ministry of Agriculture,  China.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Funding
     
    The work was supported by National Natural Science Foundation of China (32301484), Inner Mongolia Autonomous Region Natural Science Foundation General Program (2025MS03136), Grassland Talents Program of Inner Mongolia Autonomous Region, Scientific Research Foundation for Advanced Talents by Inner Mongolia  Agricultural University (NDYB2022-51), The First-Class Discipline Scientific Research Program of Inner Mongolia (IMAUCXQJ2023015), Capacity Building Project of the National Forestry and Grassland Engineering Technology Research Center for the Breeding of Drought- and Cold-Tolerant Grass Varieties in Northern China (BR221029, BR251016), 2023 National Center of Pratacultural Technology Innovation (under preparation) Major Innovation Platform Construction Project (CCPTZX2023B0701), National Key R and D Program-Intergovernmental International Science and Technology Innovation Cooperation: China-Mongolia Joint Research Project (2024YFE0113100) and Scientific Research Funding for Universities Directly under the Inner Mongolia Autonomous Region (BR22-12-07).

    CONFLICT OF INTEREST

    The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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