Effect of exogenous nitrate addition on anthocyanin accumulation in the main stem and branches of Mikania micrantha

doi:10.17521/cjpe.2025.0287

Abstract

Abstract:

Aims To investigate the effects of nitrate concentration on anthocyanin accumulation and spatial distribution in Mikania micrantha, and to reveal molecular mechanisms of adaptation to nitrogen fluctuations through secondary metabolic regulation.

Methods Controlled experiments with six nitrate nitrogen (NO₃^--N) concentrations (0, 0.5, 5, 10, 20, 40 mmol·L^-1) were conducted to measure anthocyanin content and spatial distribution in main stems and branches, combined with transcriptome analysis of key metabolic pathway genes and transcription factors.

Important findings The results show that under low nitrogen conditions (0-0.5 mmol·L^-1NO₃^--N), both the main stems and branches accumulated substantial anthocyanins to enhance stress resistance. By contrast, in treatments with medium-to-high nitrogen conditions (5-40 mmol·L^-1NO₃^--N), anthocyanin content in the main stems significantly decreased (by 43.1%-73.7%), turning them light green, while the branches remained red but with reduced anthocyanin levels, indicating a “main stem growth-branch defense” allocation strategy. Transcriptomic analysis revealed that 5 mmol·L^-1NO₃^--N suppressed the expression of key anthocyanin biosynthesis genes (PAL, CHS, CHI, F3′H, ANS, UGFT) in the main stems (downregulated by 39.2%-99.9%), while upregulating lignin synthesis genes (HCT1, CCoAOMT) and terpenoid metabolism genes (TPS11, TPS7). Additionally, nitrate modulated the expression of transcription factors (MYB (HHO₂), bHLH (PIF3)) and sucrose metabolism genes (SPS1), influencing the secondary metabolic network. This study demonstrates that M. micranthadynamically regulates anthocyanin allocation between main stems and branches, potentially to balance growth and stress resistance. This adaptive strategy might be a key mechanism contributing to its successful invasion in environments with fluctuating resource availability.

Key words: Mikania micrantha, nitrate nitrogen, anthocyanin, transcriptional regulation, invasion mechanism

CAI Min-Ling, CHEN Ming-Hao, KE Wei-Qian, PENG Chang-Lian. Effect of exogenous nitrate addition on anthocyanin accumulation in the main stem and branches of Mikania micrantha[J]. Chin J Plant Ecol, 2026, 50(6): 1370-1379.

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URL: https://www.plant-ecology.com/EN/10.17521/cjpe.2025.0287

https://www.plant-ecology.com/EN/Y2026/V50/I6/1370

Figures/Tables 7

Table 1 Primer sequences for internal gene and target gene

基因名 Gene name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
18s	GTCGGGGGCATTCGTATTTC	CGGCATCGTTTATGGTTGAG
PAL	CACGGTGGCAACTTTCAAGG	AGCTCGGAAAACTGAGCGAA
CHS	TTGGGCATCGACGATTGGAA	GCCGAAGAGTGTCGCATTTC
CHI	TATGCCGAAGAACCCGCTAC	AGCCCTGCAGCATAAACCTT
F3′H	ACACGAGATGGTGGCAAGAG	GTGTTTGAGTTCACCACGGC
DFR	ACACACCTGGCTTGTCCAAA	AGGAGTAGCGACTGCAAACC
ANS	ACCATTTGGCCCTCTAAGCC	AGTCTCCCCTCCTCCAATCC
UGFT	GGTTGAGAATCCAGGGTGGG	CATTCGATGCGACAGCCTTG
LOG	TGGGCTCAACTTGGCATTCA	TTCCGGACCAACACCTTAGC

Fig. 1 Differences in anthocyanin content in epidermal cells of main stem and branch of Mikania micrantha in treatments at different nitrate nitrogen concentrations. 0, 0.5, 5, 10, 20 and 40 indicate nitrate nitrogen concentration (mmol·L-1).

Table 2 Results (F and p) from two-way ANOVA for the effects of nitrate nitrogen (NO3--N) concentration, position, and their interaction on anthocyanin content and the expression levels of anthocyanin biosynthetic genes

变量 Variable	硝态氮浓度 NO₃^--N concentration	部位 Position	硝态氮浓度×部位 NO₃^--N concentration × position
花色素苷含量 Anthocyanin content (mmol·g⁻¹)	50.48 (0.000)	172.32 (0.000)	12.73 (0.000)
PAL基因表达量 PAL gene relative expression	67.54 (0.000)	36.68 (0.000)	37.24 (0.000)
CHS基因表达量 CHS gene relative expression	278.80 (0.000)	18.12 (0.000)	15.18 (0.000)
CHI基因表达量 CHI gene relative expression	350.85 (0.000)	40.08 (0.000)	79.09 (0.000)
F3′H基因表达量 F3′H gene relative expression	123.09 (0.000)	20.38 (0.000)	22.26 (0.000)
DFR基因表达量 DFR gene relative expression	1.88 (0.186)	23.23 (0.000)	2.31 (0.083)
ANS基因表达量 ANS gene relative expression	261.72 (0.000)	8.01 (0.000)	7.98 (0.000)
UGFT基因表达量 UGFT gene relative expression	131.03 (0.000)	20.84 (0.000)	22.99 (0.000)

Fig. 2 Changes in the anthocyanin content of main stem and branch of Mikania micrantha under different nitrate nitrogen concentrations (mean ± SE, n = 5). Different letters indicate significant differences among nitrate nitrogen concentrations for each organ (main stem or branch) (p < 0.05). ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, p ≥ 0.05.

Fig. 3 Analysis of transcription factors in the main stem of Mikania micrantha under two nitrate nitrogen treatments (0 and 5 mmol·L-1 NO3--N). A, Enrichment analysis of transcription factor gene families. B, Heatmap of transcription factor expression. A1-A3 and C1-C3 represent three biological replicate samples under 0 and 5 mmol·L-1 NO3--N treatment group, respectively. * indicate differentially expressed genes (DEGs) (p < 0.05).

Fig. 4 Heatmap of genes related to anthocyanin biosynthesis in the main stem of Mikania micrantha under two nitrate nitrogen treatments (0 and 5 mmol·L-1 NO3--N). A1-A3 and C1-C3 represent three biological replicate samples under 0 and 5 mmol·L-1 NO3--N treatment group, respectively. * indicate differentially expressed genes (DEGs) (p < 0.05).

Fig. 5 Analysis of the expression levels of anthocyanin synthesis related genes in the main stem and branches of Mikania micrantha under different concentrations of nitrate nitrogen (NO3--N) (mean ± SE, n = 5). Different letters indicate significant differences among NO3--N concentrations for each organ (main stem or branch) (p < 0.05). ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, p ≥ 0.05.

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