Unveiling the regulatory mechanisms of nodules development and quality formation in Panax notoginseng using multi-omics and MALDI-MSI

Abstract

Introduction: Renowned for its role in traditional Chinese medicine, Panax notoginseng exhibits healing properties including bidirectional regulatory effects on hematological system diseases. However, the presence of nodular structures near the top of the main root, known as nail heads, may impact the quality of the plant's valuable roots.

Objectives: In this paper, we aim to systematically analyze nail heads to identify their potential correlation with P. notoginseng quality. Additionally, we will investigate the molecular mechanisms behind nail head development.

Methods: Morphological characteristics and anatomical features were analyzed to determine the biological properties of nail heads. Active component analysis and MALDI mass spectrometry imaging (MALDI-MSI) were performed to determine the correlation between nail heads and P. notoginseng quality. Phytohormone quantitation, MALDI-MSI, RNA-seq, and Arabidopsis transformation were conducted to elucidate the mechanisms of nail head formation. Finally, protein-nucleic acid and protein-protein interactions were investigated to construct a transcriptional regulatory network of nodule development and quality formation.

Results: Our analyses have revealed that nail heads originate from an undeveloped lateral root. The content of ginsenosides was found to be positively associated with the amount of nail heads. Ginsenoside Rb₁ specifically accumulated in the cortex of nail heads, while IAA, tZR and JAs also showed highest accumulation in the nodule. RNA-seq analysis identified PnIAA14 and PnCYP735A1 as inhibitors of lateral root development. PnMYB31 and PnMYB78 were found to form binary complexes with PnbHLH31 to synergistically regulate the expression of PnIAA14, PnCYP735A1, PnSS, and PnFPS.

Conclusion: Our study details the major biological properties of nodular structures in P. notoginseng and outlines their impact on the quality of the herb. It was also determined that PnMYB31- and PnMYB78-PnbHLH31 regulate phytohormones and ginsenosides accumulation, further affecting plant development and quality. This research provides insights for quality evaluation and clinical applications of P. notoginseng.

Keywords: Ginsenoside; MALDI-MSI; Nodular structure; Panax notoginseng; Transcriptional regulation.

Graphical abstract

Fig. 1

Morphological and micrological features of nail heads in P. notoginseng. (A) P. notoginseng and nail heads on the main root. Nail heads were pointed out by black arrows. (B) P. notoginseng of different types. (C) Effect of root weight, root shape, growth year, transplantation, procession and era on number of nail heads. (D) Micrological features of nail head. (E) Micrological features of main root. (F-J) Developmental anatomy during formation of nail head. (F) Cells restoring meristem ability in cambium and phloem. Oval-labelled cells showed a thickened cytoplasm and enlarged nucleus. (G-H) Lateral root primordium-like structure in nail head. (I) The apex of primordium-like structure. Oval-labelled cells showed a thicker cell wall. (J) Micrological structure of mature nail heads. Ck, cork, Cx, cortex, Rc, resin canel, Ph, phloem, Cm, cambium, Xy, xylem, V, vessel, P, primordium. Student’s t-test was performed for comparison between two groups, *, P < 0.05, and **, P < 0.01. Variance test was performed for comparison more than two groups, with different lowercase letters indicating significant difference between groups (P < 0.05).

Fig. 2

Active component composition associated with nail head of P. notoginseng. (A) Determination of 16 ginsenosides in PRT, PLR and PNH. (B) Determination of 16 ginsenosides in P. notoginseng possessing different amounts of nail heads. (C) Content of PPD-type and PPT-type ginsenosides in PMN and PLN. (D) Determination of dencichine in PRT, PLR and PNH. (E) Determination of dencichine in P. notoginseng possessing different amounts of nail heads. (F) MALDI-MSI of ginsenoside Rb₁. ([M + K]⁺, 1147.60) in lateral root and nail head in P. notoginseng. The primordium boundary was represented in the black curve. Student’s t-test was performed for comparison between two groups, *, P < 0.05, and **, P < 0.01. Variance test was performed for comparison more than two groups, with different lowercase letters indicating significant difference between groups (P < 0.05).

Fig. 3

Dynamic distribution of endogenous phytohormoyne during nail head formation. (A1-A2) MALDI-MSI of endogenous phytohormone in PRT, PLR and PNH of different developing stages. (A3) The sampling diagram of PRT, PLR and PNH groups. (B) Determination of endogenous phytohormone in PRT, PLR and PNH (n = 10).

Fig. 4

Screening and functional verification of PnIAA14 and PnCYP735A1. (A) Expression pattern of DEGs related to phytohormone in transcriptome. (B) qRT-PCR of DEGs related to auxin signal transduction and zeatin biosynthesis (n = 3). (C) qRT-PCR of mutant plants transformed with PnIAA14 and PnCYP735A1 (n = 3). (D) Phenotype of wild type, mutants and mutants transformed with target genes. (E) Numbers of lateral roots in wild type, mutants and mutants transformed with target genes (n = 16).

Fig. 5

Transcriptional regulation of nail head formation and ginsenosides accumulation. (A) Interaction between PnMYB31, PnMYB78, PnbHLH31 and proPnFPS, proPnSS, proPnIAA14, proPnCYP735A1 in vivo (n = 3). Student’s t-test was performed, *, P < 0.05, and **, P < 0.01. (B) Structural schematics of partial promotor sequences containing MBSⅡG domain or G-box. (C) Y1H assay between PnMYB31, PnMYB78, PnbHLH31 and PnFPS, PnSS, PnIAA14, PnCYP735A1. Grey triangles represented dilution factor of the yeast concentration. (D) EMSA between PnMYB31, PnMYB78, PnbHLH31 and PnFPS, PnSS, PnIAA14, PnCYP735A1. (E) BiFC assay between PnMYB31/PnMYB78 and PnbHLH31. Horizontal line on the right bottom represented 10 μm. (F) LCI assay between PnMYB31/PnMYB78 and PnbHLH31. Horizontal line on the right bottom represented 1 cm.

Fig. 6

A probable regulatory mechanism model for nail head formation in P. notoginseng.