Camellia oil tree, a perennial evergreen shrub or small tree indigenous to China, is a vital woody oilseed species. Its seed-derived oil is widely recognized as a premium edible oil. Crucially, this species predominantly thrives in hilly regions, effectively alleviating arable land pressure and mitigating competition with staple crops, thereby contributing to national food security. The escalating impacts of global climate change have established drought as a critical constraint on Camellia oil tree growth and productivity (Tan, 2023; Zhou et al., 2023). Previous studies showed that glutamate content in rhizosphere soil was closely related to the degree of drought (Yuan et al., 2024). Glutamate (Glu), a multifunctional metabolic product, serves as a critical amino acid in plant systems. As a central component of nitrogen metabolism, Glu and its derivatives directly participate in synthesizing and metabolizing nitrogen-containing compounds, including carbohydrates, fatty acids, and other amino acids, thereby bridging carbon and nitrogen metabolic pathways (Liao et al., 2022). Over 95% of plant NH₄⁺—derived from root absorption or nitrate reductase (NR)- and nitrite reductase (NiR)-mediated NO₃^– reduction—is assimilated via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, where Glu acts as a pivotal intermediary (Fortunato et al., 2023). This underscores its indispensable role in nitrogen metabolism in higher plants.

Amino acids, including Glu, are increasingly incorporated into organic fertilizers to mitigate abiotic stresses. Functioning as a primary metabolite, nitrogen source, and signaling molecule, Glu contributes to plant adaptation under stress conditions (Liao et al., 2022; Qiu et al., 2020). For instance, foliar application of 4 mmol/L Glu under drought stress enhances wheat dry matter accumulation, photosynthetic efficiency, and grain quality while minimizing yield loss (Li et al., 2024). When utilized as a nitrogen source in rice, Glu is rapidly metabolized into compounds like glutamine rather than accumulating (Kan et al., 2017). Furthermore, Glu bolsters plant tolerance to cold, salinity, cadmium, and arsenic by improving photosynthetic capacity and antioxidant system performance (Asgher et al., 2022; Franzoni et al., 2022; Gai et al., 2020; Lee et al., 2021). However, excessive Glu accumulation under NH₄⁺ stress can impair the tricarboxylic acid (TCA) cycle, suppressing plant growth (Wang et al., 2020). Beyond stress adaptation, Glu influences microbial community dynamics. In strawberry anthosphere studies, Glu supplementation drastically altered microbial composition, with Streptomycetaceae dominating 99.9% of the community by weeks 6 and 8 post-treatment (Kim et al., 2021). Amino acids, as vital nitrogen sources for plants and microbes, trigger competitive uptake in soil systems: microbes rapidly assimilate these compounds, though plants exhibit superior absorption efficiency (Jones et al., 2005). Similarly, organic matter enriched with phenylalanine or leucine enhances humus formation via bacterial activity (Zheng et al., 2021).

While Glu research has predominantly focused on plant growth regulation, shifts in rhizosphere microbiomes remain underexplored. This gap is particularly significant given that root-associated microbial communities— often termed the plant second genome, play essential roles in supporting plant growth and development through multiple mechanisms (Berendsen et al., 2012). Advancing our understanding of how rhizosphere microbes mediate plant adaptation to abiotic stress is pivotal for enhancing future resilience to extreme environmental conditions.

Current analytical methodologies for assessing microbial dynamics are constrained by critical limitations. Although relative quantification sequencing has been extensively employed to link shifts in taxonomic composition with environmental gradients (Props et al., 2017; Wang et al., 2024), this approach yields a partial understanding of microbial ecology. Emerging evidence demonstrates that apparent increases in relative abundance of specific taxa might represent not biological proliferation but rather the competitive suppression of coexisting species. Conversely, absolute quantification sequencing enables precise detection of subtle microbial population fluctuations at heightened resolution, thereby providing superior capacity to monitor community-level adaptations to environmental stressors (Maghini et al., 2024; Tkacz et al., 2018). This methodological dichotomy highlights the imperative for incorporating absolute quantification frameworks to resolve complex plant-microbe interaction mechanisms under abiotic stress conditions.

Previous studies by our group have demonstrated that Glu content in Camellia oil tree rhizosphere soil rises with escalating drought severity. However, whether this drought-induced Glu accumulation induces structural shifts in the rhizosphere microbiome remains unresolved. In this study, we integrate absolute and relative quantification data to analyze the effects of exogenous Glu on the rhizosphere microbial community of Camellia oil tree, thereby elucidating its regulatory mechanisms on microbial composition and function. These findings aim to establish a theoretical foundation for development and utilization of rhizosphere microorganisms in the future.

This study demonstrated that exogenous Glu application under drought conditions predominantly altered nitrogen dynamics in the rhizosphere soil of Camellia oil tree, with DON being the most significantly impacted fraction. Bacterial community diversity trends were largely consistent between absolute and relative quantification methodologies. However, relative abundance quantification failed to reflect the pronounced inter-treatment disparities in Acidobacteriota and Pseudomonadota abundances. Functional prediction analysis further revealed that the observed inter-treatment differences were principally linked to nitrogen fixation, nitrification, and denitrification processes.

Discrepancies were observed between absolute and relative quantitative results for alpha and beta diversity. Relative quantification largely aligned with absolute quantification in treatments exhibiting pronounced differences, such as between G5 and G10 (Figures 2a,b). However, it inadequately resolved subtle differences between treatments with smaller variations, such as G0, G1, and G2. Additionally, relative quantification failed to capture changes in total bacterial community abundance under G5 and G10 treatments in analyses of community diversity (Figure 3a). These findings suggest that absolute quantification may be preferable when analyzing microbial datasets with minor inter-treatment differences.

This study demonstrated that increasing Glu application induced progressive soil acidification—a finding contrary to previous reports suggesting rhizosphere alkalization from exogenous arginine, proline, glutamine, or Glu due to ammonia accumulation (Liu et al., 2023). This discrepancy may stem from differences in rhizosphere microbiota between Camellia oil tree and model plants (e.g., Arabidopsis) or cultivation substrates (soil vs. artificial media). The acidic red soil used in this study, characteristic of subtropical monsoon regions, is non-calcareous with lower CaCO3 content compared to temperate calcareous soils. Consequently, its reduced acid-buffering capacity results in heightened sensitivity to nitrogen inputs and predisposition to acidification (Dong et al., 2025; Hao et al., 2022). Additionally, the molecular structure of Glu (two carboxyl groups and one amino group) may further exacerbate acidification through proton release during dissociation under acidic conditions (Bolan and Hedley, 2003). Despite decreasing pH trends, DON, NH₄⁺-N, and NO₃^–-N concentrations increased proportionally with Glu dosage. As a plant-available nitrogen source, Glu supplementation through irrigation directly elevates soil nitrogen levels. However, TN content showed no significant variation between G5 and G10 treatments, potentially due to counterbalancing trends among other nitrogen pools—a phenomenon requiring further investigation.

Soil enzymes serve as critical biocatalysts in soil ecosystems, with their activity closely associated with nutrient availability (Aransiola et al., 2022). CAT and βG are centrally involved in the soil carbon cycle. CAT mitigates hydrogen peroxide accumulation, reducing its toxic effects on soil organisms while catalyzing biochemical reactions linked to microbial decomposition and metabolic processes. βG, a key component of the cellulase family, primarily facilitates the degradation of lignin and cellulose (Chen et al., 2022; Kou et al., 2022). Consistent with prior studies, CAT and βG activities decreased under low soil moisture but increased with exogenous nitrogen inputs (Bogati and Walczak, 2022; Dong et al., 2022). This phenomenon may be attributed to Glu’s role as a free amino acid, which is readily assimilated by soil microorganisms, enhancing nitrogen and carbon availability and stimulating microbial activity. Ure and LAP are integral to soil nitrogen dynamics: Ure regulates nitrogen availability through urea hydrolysis, while LAP releases leucine via protein hydrolysis (Chen et al., 2018). However, this study observed reduced Ure and LAP activities with increasing Glu concentrations, contrasting previous findings (Chen et al., 2018; Dong et al., 2022). This divergence likely stems from differences in nitrogen sources—high-purity Glu here versus mixed-form fertilizers in prior studies. Furthermore, direct rhizospheric application of dissolved Glu may have promoted preferential root uptake, limiting substrate availability for Ure and LAP and thereby suppressing enzymatic activity. Phy and ACP activities remained stable across treatments, likely due to the absence of phosphorus in Glu, precluding direct influence on soil phosphorus cycling.

To date, no studies have explicitly examined the effects of exogenous Glu on plant rhizosphere microbiota. This study observed increased Shannon and Chao1 index with escalating Glu dosage. SEM revealed that Glu exerted positive effects on both soil nutrients and bacterial alpha diversity, while soil nutrients demonstrated a negative regulatory effect on bacterial alpha diversity. A meta-analysis indicates that exogenous N application under field conditions reduces soil microbial Shannon and Chao1 index (Wang C. et al., 2018). This divergence may be attributable to three interrelated mechanisms: first, interference from co-occurring nitrogen sources. Prior meta-analyses predominantly derived data from fertilized agroecosystems or forests, where nitrogen inputs arise from anthropogenic activities or sustained organic matter decomposition (Wang C. et al., 2018). In this study, soil was sourced from an unfertilized Camellia oil tree plantation with surface litter removed, yielding nutrient-poor conditions. Consequently, limited nitrogen availability may have amplified microbial proliferation following Glu supplementation. Second, disparate research objectives: agricultural and forest studies typically prioritize yield enhancement and economic trait optimization through nitrogen addition, whereas this work focused on drought resilience—a distinction that likely influenced microbial community responses. Thirdly, the application of Glu concentrations significantly altered soil pH, which may negatively regulate bacterial alpha diversity through modified soil nutrient profiles. This finding suggest that Glu may play a dual role in the soil microbial environment. In addition to functioning as a nitrogen source, Glu likely contributes to enhanced bacterial diversity by fulfilling microbial nutritional demands through amino acid metabolic pathways. However, direct experimental evidence supporting the role of Glu as an amino acid modulator of microbial diversity remains lacking. Future work will focus on disentangling the causal relationships among Glu, pH, and bacterial community structure.

Concurrently, we hypothesize that beyond serving as a nitrogen source, Glu may directly promote bacterial diversity by meeting microbial nutritional requirements through amino acid metabolic pathways. However, this study does not provide direct evidence supporting the role of Glu as an amino acid in modulating bacterial diversity. Further experimental investigations are planned to elucidate the causal relationships among Glu, pH, and bacterial diversity.

This study identified Acidobacteriota, Pseudomonadota, Chloroflexota, and Actinomycetota as the dominant rhizosphere bacterial phyla, aligning with prior research (Wang C. et al., 2018; Weng et al., 2023). Glu supplementation significantly increased the absolute abundance of Acidobacteriota and Pseudomonadota. Acidobacteriota, ubiquitous across diverse ecosystems, exhibit metagenomic-level functional traits linked to amino acid and carbohydrate metabolism, indirectly modulating plant growth. Additionally, they demonstrate enzymatic activity in degrading cellulose, dextran, starch, and peptidoglycan (Gonçalves et al., 2024). Soil pH is a critical regulator of Acidobacteriota dynamics, with their abundance inversely correlated with pH (Chen et al., 2024). The observed abundance increase may stem from Glu-induced soil acidification via hydrogen ion release during hydrolysis. Furthermore, Acidobacteriota’s strong association with nitrogen cycling suggests Glu-mediated nitrogen enrichment likely enhanced their competitiveness (Gonçalves et al., 2024). Pseudomonadota exhibited parallel abundance trends to Acidobacteriota, reflecting shared sensitivities to pH and nitrogen availability. Previous studies have established Pseudomonadota’s responsiveness to irrigation and nitrogen inputs, irrespective of nitrogen form (Zhang et al., 2014). Notably, absolute quantification revealed Glu-driven increases in total bacterial biomass across treatments, contrasting with the relative abundance trade-offs observed in compositional analyses.

Previous studies have demonstrated that nitrogen supplementation elevates soil inorganic nitrogen leaching, nitrification, nitrous oxide emissions, and denitrification (Lu et al., 2011), a pattern corroborated by this study. Notably, total bacterial abundance, trends in differentially abundant phyla, and the expression profiles of most nitrogen cycle genes peaked at the G5 treatment before declining. This implies that the benefits of Glu supplementation to soil systems are subject to diminishing returns, likely governed by a concentration-dependent threshold (Zhang et al., 2017). In this study, 5 mmol/L Glu appears to delineate this threshold, where maximal increases in nitrogen fixation-, nitrification-, and denitrification-associated gene abundances occurred. It should be noted that PICRUSt2 functional predictions depend on phylogenetic extrapolation from 16S rRNA data to annotated reference genomes (Douglas et al., 2020). In our analysis, 33% of ASVs were mapped to reference genomes with an NSTI value ≤ 0.2 (threshold: 2), while 75% of ASVs were mapped to those with an NSTI ≤ 0.5. These results suggest that a substantial proportion of the microbial community is phylogenetically closely related to sequenced taxa in reference databases. These methodological constraints must be explicitly acknowledged during ecological interpretation, particularly for nitrogen cycling processes given their enzymatic complexity, through either undetected functional contributions or distortion of predicted gene abundance ratios. Its accuracy exhibits reduced reliability in ecologically complex or poorly characterized ecosystems; consequently, metagenomic approaches remain the preferred methodology for detecting authentic gene abundance variations.

As an amino acid and nitrogen source, Glu can be efficiently utilized by plants; however, its cost about three–four times that of traditional nitrogen fertilizers (Table 3, data sourced from https://www.1688.com, with price fluctuations recorded as of 4/18/2025). Although less cost-effective, Glu offers advantages in water-soluble application and superior plant uptake efficiency compared to conventional nitrogen fertilizers, making it particularly suitable for rapid nitrogen supplementation during plant nitrogen deficiency.

This study offers insights into rhizosphere microbial community dynamics following root application of Glu as an exogenous N/amino acid in Camellia species (or analogous crops) in agricultural production, while also providing guidance on optimal application concentrations.

Notably, this study has several limitations. First, the study revealed a significant decrease in soil pH after Glu application, suggesting that prolonged use of Glu may accelerate soil acidification. Co-application with quicklime and other alkaline matter or implementation in alkaline soil conditions (pH > 7.5) may constitute a preferable approach. Second, the 30-day pot experiment captured short-term microbial responses, which may reflect transient adaptation fluctuations, and further temporal variations cannot be excluded. Third, the study did not assess the effects of Glu on Camellia oil tree growth. Future field-based studies should investigate the long-term impacts of Glu on rhizosphere microorganisms and plant development, clarifying whether short-term microbial fluctuations persist and whether adverse effects on plants emerge. Such work could explore broader applications of Glu in agroforestry systems, establishing a robust theoretical foundation for developing precise and sustainable agricultural practices.

This study demonstrated that Glu supplementation significantly altered soil available nitrogen dynamics, including DON, ammonium NH₄⁺-N, and NO₃^–-N. Absolute quantification revealed that escalating Glu concentrations enhanced the abundance of Acidobacteriota and Pseudomonadota, resulting in a unimodal trend of total bacterial abundance (initial increase followed by decline). The 5 mmol/L Glu treatment emerged as a critical threshold, beyond which bacterial abundance and nitrogen cycle-related gene expression in differential taxa declined. These findings collectively suggest that 5 mmol/L Glu represents a pivotal concentration influencing rhizosphere bacterial community dynamics in Camellia oil tree under drought stress.