**Background:** Phosphorus pollution from agricultural and industrial sources causes eutrophication in aquatic ecosystems. Constructed wetlands (CWs) are an efficient biological treatment method, and plant selection is critical for phosphorus removal. Myriophyllum aquaticum has demonstrated >90% phosphorus removal efficiency in CWs and can grow under varying nutrient concentrations. While the molecular mechanisms of nitrogen stress response in M. aquaticum have been studied, the transcriptome-level response to phosphorus stress was previously unknown. This study aimed to elucidate the gene regulatory networks enabling M. aquaticum to cope with both low and high phosphorus stress.
**Methods:** M. aquaticum seedlings were cultivated in containers with 25 L of 50% Hoagland solution at three phosphorus concentrations: 0.02 mM (low stress), 0.25 mM (normal control), and 5 mM (high stress), with three replicates per group. Plants were grown under a 12/12 h dark/light photoperiod at 26 ± 2°C for 14 days, with medium changed every 2 days and pH maintained at 6.0. Growth rate, root length and number, and chlorophyll a and b contents were measured. RNA was extracted from root and leaf tissues using an EASYspin Plus Complex Plant RNA Kit. Transcriptome sequencing was performed on 18 samples using the Illumina Hiseq X Ten platform (PE150 paired-end). After quality filtering, 788 million clean reads and 11.82 billion clean bases were obtained, with an average clean reads rate of 85.01% and average clean Q30 Bases Rate of 94.54%. Assembly with Trinity software yielded 31,728 unigenes (N50 = 1625, N90 = 557). DEGs were identified using DESeq2 with FDR < 0.05 and |log2FoldChange| ≥ 1. GO and KEGG enrichment analyses were performed.
**Key Results:** Low phosphorus stress significantly decreased growth rate by 4.22 times (p < 0.01) compared to normal phosphorus and 2.81 times (p < 0.05) compared to high phosphorus stress. Neither low nor high phosphorus stress significantly reduced chlorophyll a or b content (p > 0.05), though low phosphorus had greater impact. PCA showed distinct separation of gene expression profiles between stress and control groups, with roots showing greater separation than leaves (PC1 explained 74.83% of variation). In total, 2507 common DEGs were identified across all treatments. Roots had more DEGs than leaves under both stress conditions. Up-regulated genes outnumbered down-regulated genes in all groups. Under low phosphorus stress, GO enrichment showed biological processes dominated, including cellular processes, metabolic processes, and biological regulation. KEGG analysis revealed that high phosphorus stress led to more significantly enriched pathways than low phosphorus stress: 15 pathways for leaves under low stress (LL), 5 for roots under low stress (RL), 26 for leaves under high stress (LH), and 21 for roots under high stress (RH). Pathways commonly enriched across all groups included cutin, suberine and wax biosynthesis (ko00073), plant hormone signal transduction (ko04075), and circadian rhythm (ko04710). Under low phosphorus stress, photosynthesis-related DEGs were significantly enriched in leaves, while energy metabolism and transport–catabolism pathways were severely inhibited in roots. Under high phosphorus stress, no photosynthesis-related DEGs were observed in leaves, but pathways including glycosphingolipid biosynthesis-ganglio series (ko00603) and GPI-anchor biosynthesis (ko00563) were enriched. Antioxidant enzyme gene expression showed a larger decline under low phosphorus than high phosphorus stress. Phosphorus transporter gene expression increased under low phosphorus concentration.
**Clinical Implications:** This study provides the first comprehensive transcriptome-level analysis of phosphorus stress response in M. aquaticum. The findings demonstrate that M. aquaticum employs distinct gene regulation strategies for low versus high phosphorus stress, with complex interconnected networks involving photosynthesis, oxidative stress reduction, phosphorus metabolism, signal transduction, secondary metabolite biosynthesis, and energy metabolism. The plant's superior ability to regulate these pathways, particularly under high phosphorus stress, supports its use in constructed wetlands for treating phosphorus-rich wastewater. The identified genes and pathways may guide future genetic improvement of macrophytes for wastewater treatment applications. However, the authors note that conclusions drawn from statistically significant differential expression of assembled transcripts are preliminary and should be confirmed by quantitative methods such as qPCR.