Gibberellin Signaling Promotes the Secondary Growth of Storage Roots in Panax ginseng

Abstract

Gibberellins (GAs) are an important group of phytohormones associated with diverse growth and developmental processes, including cell elongation, seed germination, and secondary growth. Recent genomic and genetic analyses have advanced our knowledge of GA signaling pathways and related genes in model plant species. However, functional genomics analyses of GA signaling pathways in Panax ginseng, a perennial herb, have rarely been carried out, despite its well-known economical and medicinal importance. Here, we conducted functional characterization of GA receptors and investigated their physiological roles in the secondary growth of P. ginseng storage roots. We found that the physiological and genetic functions of P. ginseng gibberellin-insensitive dwarf1s (PgGID1s) have been evolutionarily conserved. Additionally, the essential domains and residues in the primary protein structure for interaction with active GAs and DELLA proteins are well-conserved. Overexpression of PgGID1s in Arabidopsis completely restored the GA deficient phenotype of the Arabidopsis gid1a gid1c (atgid1a/c) double mutant. Exogenous GA treatment greatly enhanced the secondary growth of tap roots; however, paclobutrazol (PCZ), a GA biosynthetic inhibitor, reduced root growth in P. ginseng. Transcriptome profiling of P. ginseng roots revealed that GA-induced root secondary growth is closely associated with cell wall biogenesis, the cell cycle, the jasmonic acid (JA) response, and nitrate assimilation, suggesting that a transcriptional network regulate root secondary growth in P. ginseng. These results provide novel insights into the mechanism controlling secondary root growth in P. ginseng.

Keywords: GID1s; Panax ginseng; cell wall biogenesis; gibberellins; phytohormones; storage root secondary growth.

Figure 1

Exogenous gibberellin (GA) treatment promotes primary growth of stems and secondary growth of roots in Panax ginseng. (A) Phenotype of 1-year-old P. ginseng plants treated with DMSO (control [Con]), 10 μM GA₃, and 100 μM paclobutrazol (PCZ) once a week for 8 weeks. Scale bar = 2 cm. (B) Measurements of shoot length and root diameter. (C) Representative images of stained stem cross-sections of P. ginseng plants treated with DMSO (Con), GA₃ and PCZ. Scale bar = 100 μm. (D) Quantification of cell length in the indicated treatments. (E) Representative images of stained root cross-sections of P. ginseng plants treated with DMSO (Con), GA₃, and PCZ. XV: xylem vessel, CZ: cambial cell layer zone, RD: resin duct cells. Scale bar = 100 μm (F) Quantification of cambium-derived cells in the XV and RD of each ray. In (B,D,F), dots, squares and triangles represent individual values. Error bars represent standard error; n = 16 (B), 20 (D), 10 (F). Different lowercase letters indicate statistically significant differences (p < 0.05; one-way analysis of variance [ANOVA], followed by Tukey’s multiple range test).

Figure 2

Phylogenetic analysis and amino acid sequence alignment of GID1 proteins. (A) Phylogenetic analysis of PgGID1A–H, AtGID1s, and OsGID1. The phylogenetic tree was constructed using the MEGA7 program. Horizontal branch lengths are proportional to the estimated number of amino acid substitutions per residue. Bootstrap values were obtained by 1000 bootstrap replicates. Pg, Panax ginseng; At, Arabidopsis thaliana; Os, Oryza sativa. (B) Topology diagram based on the predicted secondary structure of the OsGID1 protein [15]. (Blue circles indicate important residues involved in the GID1–SLR1 interaction, and red dots indicate important residues involved in OsGID1–GA and GID1–SLR1 interactions. Red stars indicate residues essential for enzymatic activity. Colored zones indicate the lid (yellow) and binding pocket (red). (C) Amino acid sequence alignment of the GID1 proteins of Arabidopsis, rice, and P. ginseng constructed using SMS (https://www.Bioinformatics.org (accessed on 1 May 2021)). (D) Subcellular localization analysis of PgGID1A–D proteins in Arabidopsis protoplasts. Full-length coding sequences of PgGID1A–D were fused to the GFP reporter gene. The nucleus was visualized using the AtARR2-RFP nuclear marker. GFP and RFP fluorescence images were merged. Scale bar = 50 μm.

Figure 3

Complementation of the Arabidopsis gid1 loss-of-function mutant via PgGID1 overexpression. (A) Rescue of the GA-insensitive dwarf phenotype of the atgid1a/c double mutant by overexpression of PgGID1A–D genes. Scale bar = 5 cm (top panel) and 0.5 cm (bottom panel). (B) Measurement of the length of shoots and siliques shown in (A). Error bars represent the standard error (n > 12). Different lowercase letters indicate statistically significant differences (p < 0.05; one-way ANOVA, followed by Tukey’s multiple range test). (C) Western blot analysis of PgGID1A–D protein levels in atgid1a/c plants. (D) Yeast two-hybrid assay. To test the interaction between PgGID1s and PgRGAs, PgGID1A–D-carrying pGBKT7 constructs (bait) were co-expressed with PgRGA1–5-harboring pGADT7 constructs (prey) in yeast cells, which were grown on media (-LTH) supplemented with or without 100 nM GA₃.

Figure 4

Transcriptome profiling of P. ginseng roots treated with or without GA. (A) Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) identified by comparison of GA- and DMSO-treated root samples. GO terms of level 3 (yellow bars) and level 5 (black bars), with EASE score < 0.01, were selected (left panel). The number of up-regulated genes (red) and down-regulated genes (blue) categorized under the enriched GO terms are shown in the right panel. (B) Enrichment plot for the responses to ABA (GO: 0009737) and JA (GO: 0009753) in the transcriptome data of DMSO- and GA-treated root samples. In the enrichment plot, the red dotted line 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 DMSO-treated root samples with red color gradient, and negative values indicate genes down-regulated in DMSO-treated root samples.

Figure 5

Functional enrichment of cell wall biogenesis in P. ginseng root in response to GA and the function-related transcriptional network. (A) Enrichment plot for the plant-type secondary cell wall biogenesis (GO: 0009834), and an expression heatmap of 55 genes related to this pathway (FDR = 0.0). (B) Transcriptional network of up-regulated genes related to cell wall biogenesis (57 genes), cell cycle and division (120 genes), cell growth (54 genes), response to JA (42 genes), and nitrate assimilation (13 genes). In the network, red lines indicate the connection of genes between cell wall biogenesis and other functional categories.