Nitrate enhances the secondary growth of storage roots in Panax ginseng

Abstract

Background: Nitrogen (N) is an essential macronutrient for plant growth and development. To support agricultural production and enhance crop yield, two major N sources, nitrate and ammonium, are applied as fertilizers to the soil. Although many studies have been conducted on N uptake and signal transduction, the molecular genetic mechanisms of N-mediated physiological roles, such as the secondary growth of storage roots, remain largely unknown.

Methods: One-year-old P. ginseng seedlings treated with KNO₃ were analyzed for the secondary growth of storage roots. The histological paraffin sections were subjected to bright and polarized light microscopic analysis. Genome-wide RNA-seq and network analysis were carried out to dissect the molecular mechanism of nitrate-mediated promotion of ginseng storage root thickening.

Results: Here, we report the positive effects of nitrate on storage root secondary growth in Panax ginseng. Exogenous nitrate supply to ginseng seedlings significantly increased the root secondary growth. Histological analysis indicated that the enhancement of root secondary growth could be attributed to the increase in cambium stem cell activity and the subsequent differentiation of cambium-derived storage parenchymal cells. RNA-seq and gene set enrichment analysis (GSEA) revealed that the formation of a transcriptional network comprising auxin, brassinosteroid (BR)-, ethylene-, and jasmonic acid (JA)-related genes mainly contributed to the secondary growth of ginseng storage roots. In addition, increased proliferation of cambium stem cells by a N-rich source inhibited the accumulation of starch granules in storage parenchymal cells.

Conclusion: Thus, through the integration of bioinformatic and histological tissue analyses, we demonstrate that nitrate assimilation and signaling pathways are integrated into key biological processes that promote the secondary growth of P. ginseng storage roots.

Keywords: Hormone; Nitrate; Panax ginseng; Root secondary growth.

Graphical abstract

Fig. 1

Potassium nitrate (KNO₃) treatment promotes shoot primary growth and root secondary growth in P. ginseng. (A) Measurement of the diameter of one-year-old P. ginseng roots treated with a mock control (Con), 5 mM ammonium chloride (NH₄Cl) or potassium nitrate (KNO₃) once a week for 8 weeks. Error bars represent standard error (n = 10). Different lowercase letters indicate statistically significant differences P < 0.05; one-way analysis of variance [ANOVA], followed by Tukey's multiple range test). (B) Phenotype of 1-year-old P. ginseng plants treated with a mock control (Con) or 5 mM potassium nitrate (KNO₃) once a week for 8 weeks. Scale bar = 2 cm. (C) Measurement of shoot length and root diameter of Fig. 1B. Dots in graphs represent individual values. Error bars represent standard error; n = 10 (∗P < 0.05, ∗∗P > 0.01, The significance of the difference was analyzed by t-test method.).

Fig. 2

Histological sections of KNO₃-treated shoots and roots of P. ginseng. (A) Representative stem images of stained stem cross-sections of P. ginseng plants treated with DMSO (Con) or 5 mM KNO₃. Scale bar = 100 μm. (B) Quantification cell length in (A) was measured using ImageJ software. (C) Representative root images of stained stem cross-sections of P. ginseng plants treated with DMSO (Con) or 5 mM KNO₃. XV: Xylem vessel, CZ: Cambial cell layer zone, RD: Resin duct cells. Scale bar = 100 μm. (D) Quantification of cambium-derived cells in XV and RD of each ray. Dots in graphs represent individual values. Error bars represent standard error; n = 25 (B), 11 (D). (∗P < 0.05, ∗∗P > 0.01, The significance of the difference was analyzed by t-test method.).

Fig. 3

KNO₃ treatment enhances the cambial stem cell activity of ginseng roots. (A, B) Phenotype of the cambial stem cells of 1-year-old P. ginseng roots treated with control (A) and 5 mM KNO₃ (B) for two and eight weeks. Scale bar = 100 μm. (C) The numbers of cambial stem cells in the cambial cell layer zone (CZ). Error bars represent standard error (n = 15). Different lowercase letters indicate statistically significant differences P < 0.05; one-way ANOVA, followed by Tukey's multiple range test.

Fig. 4

Transcriptome profiling of P. ginseng roots grown with or without KNO₃. (A) MA plot of differential expression between mock-and KNO₃-treated samples. Blue dots and bar graph represent the either up-(4030) and down-(4011) regulated genes with q < 0.05 and ≥ |1.5|-fold change. (B) Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) identified by comparison of control and KNO₃-treated root samples. GO terms in biological process of level 3 and level 5, with EASE score <0.01, were selected (left panel). The number of up-regulated genes (red) and down-regulated genes (green) categorized under the enriched GO terms are shown in the right panel. (C) Enrichment plot for a nitrate assimilation (GO:0042128) in the transcriptome data of control and KNO₃-treated ginseng root samples. In the enrichment plot, the red dotted line (leading-edge subset) represents the gene subset that made the largest contribution to the enrichment score (ES) (false discovery rate [FDR] < 0.05). The ranking list metric in the plot measures the correlation between a gene and the plant phenotype. In the ranking list, positive values indicate genes up-regulated in mock-treated root samples with red color gradient, and negative values indicate genes down-regulated in mock-treated root samples. (D) Expression heatmap of the leading-edge subset genes (red line) contained in the nitrate assimilation related GO term.

Fig. 5

Functional enrichment of auxin-activated signaling pathway and brassinosteroid homeostasis related terms in the nitrate-mediated secondary growth promotion of P. ginseng root. (A, B) Enrichment plot for the auxin-activated signaling pathway (A, GO:0009734, FDR = 0.086), and an expression heatmap of leading-edge subset genes related to this pathway (B). (C) Enrichment plot for the brassinosteroid homeostasis (GO:0010268, FDR = 0.021), and an expression heatmap of leading-edge subset genes. Red lines indicate the leading-edge subset genes in the GSEA.

Fig. 6

Protein-protein interaction (PPI) network analysis of DEGs reveals other important pathways for nitrate-mediated secondary growth of ginseng roots. (A) PPI network analysis of DEGs. (B, C) Enrichment plot for the jasmonic acid mediated signaling pathways (B, GO:0009867, FDR = 0.061) and ethylene biosynthetic process (C, GO:0009693, FDR = 0.061), and expression heatmaps of leading-edge subset genes. Red lines indicate the leading-edge subset genes in the GSEA.

Fig. 7

Nitrate negatively regulates carbohydrate biosynthetic process and storage parenchymal cell development in ginseng roots. (A, B) Enrichment plot for the carbohydrate biosynthetic process (A, GO:0016051, FDR = 0.086), and an expression heatmap of leading-edge subset genes related to this pathway (B). (C, D) Starch granule development in storage parenchyma cells of P. ginseng tap roots grown with (C) or without (D) 5 mM KNO₃. (E) A schematic model for nitrate-mediated transcriptional network for promoting secondary growth of P. ginseng storage root (The model was created with BioRender.com).