Increasing Expression of PnGAP and PnEXPA4 Provides Insights Into the Enlargement of Panax notoginseng Root Size From Qing Dynasty to Cultivation Era

Abstract

Root size is a key trait in plant cultivation and can be influenced by the cultivation environment. However, physical evidence of root size change in a secular context is scarce due to the difficulty in preserving ancient root samples, and how they were modified during the domestication and cultivation stays unclear. About 100 ancient root samples of Panax notoginseng, preserved as tribute in the Palace Museum (A.D. 1636 to 1912, Qing dynasty), provided an opportunity to investigate the root size changes during the last 100 years of cultivation. The dry weight of ancient root samples (~120 tou samples, tou represents number of roots per 500 g dry weight) is 0.22-fold of the modern samples with the biggest size (20 tou samples). Transcriptome analysis revealed that PnGAP and PnEXPA4 were highly expressed in 20 tou samples, compared with the 120 tou samples, which might contribute to the thicker cell wall and a higher content of lignin, cellulose, and callose in 20 tou samples. A relatively lower content of dencichine and higher content of ginsenoside Rb₁ in 20 tou samples are also consistent with higher expression of ginsenoside biosynthesis-related genes. PnPHL8 was filtrated through transcriptome analysis, which could specifically bind the promoters of PnGAP, PnCYP716A47, and PnGGPPS3, respectively. The results in this study represent the first physical evidence of root size changes in P. notoginseng in the last 100 years of cultivation and contribute to a comprehensive understanding of how the cultivation environment affected root size, chemical composition, and clinical application.

Keywords: GPI-anchored; Panax notoginseng; cell wall; cultivation; expansin; root size.

Figure 1

Morphological features of P. notoginseng in Qing dynasty and modern times. (A) P. notoginseng in Qing dynasty, relic number, Gu00172482-8/9. (B) P. notoginseng of different root sizes (20 tou, 40 tou, 60 tou, 80 tou, 120 tou, and >120 tou; tou is the number of roots per 500 g). (C) Length, diameter, and weight of P. notoginseng in Qing dynasty (n = 30) and in modern times (n = 20). Data are shown in box plot, and the different letters indicate values that vary significantly at P < 0.05 (one-way ANOVA).

Figure 2

Different tissues and cell morphology in P. notoginseng. SRW was an abbreviation of small root weight, while LRW represented large root weight. (A) Microscope morphology of different tissues including cork, cortex, phloem, xylem, and vessel, and comparison of the cell wall thickness. (B) Comparison of the vessel diameter of P. notoginseng with different root weights. (C) Determination of lignin and callose in P. notoginseng with different root weights. The thickness of 10 cell walls in one field of view were measured and averaged, and then in accordance with this, a total of six fields of view from five biological replicates were obtained for t-test. Measurement of the vessel diameters was same as that of the cell wall thickness, while the diameter of seven vessels was measured and averaged. Asterisks denote Student's t-test significance: *P < 0.05 and **P < 0.01.

Figure 3

Expression pattern of EXPs and EXTs in LRW and SRW samples. EXPA, alpha-expansin; EXPB, beta-expansin; EXT, extension. Asterisks denote student's t-test significance: *P < 0.05.

Figure 4

Expression pattern of genes related to biosynthesis of ginsenosides and content of active components. (A) Expression pattern of genes related to biosynthesis of ginsenosides in LRW and SRW groups. (B) Content of active components in LRW and SRW groups. AACT, acetyl-CoA C-acetyltransferase; HMGS, hydroxymethylglutaryl-CoA synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; HMGR, hydroxymethylglutaryl-CoA reductase; MVK, mevalonate kinase; MVAP, mevalonate-5-phosphate; PMK, phosphomevalonate kinase; MVAPP, mevalonate-5-pyrophosphate; MVD, diphosphomevalonate decarboxylase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPS, geranyl pyrophosphate synthase; GPP, geranyl pyrophosphate; FPS, farnesyl diphosphate synthase; FPP, farnesyl diphosphate; GGPPS, geranylgeranyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; DS, dammarenediol-II synthase; CYP, cytochrome P450 proteins; UGT, UDP-glycosyltransferase; GS, ginsenoside; NG, notoginsenoside. Genes obtained * on upper right were reported to be functional. Genes in red tag had statistical significance. Asterisks denote Student's t-test significance: *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

PnPHL8 binds with the promoter sequence of PnGAP, PnCYP716A47, and PnGGPPS3. (A) Structural schematics of baits and prey in yeast one-hybrid (Y1H) assay. (B) Y1H assay between PnPHL8 and PIBS domains as well as MBS domains of proPnGAP (promoter of PnGAP), proPnEXPA4 (promoter of PnEXPA4), proPnGGPPS3 (promoter of PnGGPPS3), proPnCYP716A47 (promoter of PnCYP716A47), proPnCYP716A53v2 (promotor of PnCYP716A53v2), and proPnFPS (promotor of PnFPS). Gray triangles represent dilution factor of the yeast concentration, while p53 served as a positive control. (C) EMSA among PnPHL8, proPnGAP, proPnCYP716A47, and proPnGGPPS3. GAP, glycosylphosphatidylinositol-anchored protein; PHL, phosphate starvation response transcription factor like; AbA, aureobasidin.

Figure 6

Probable molecular mechanism model for cultivation influencing root architecture and chemical component of P. notoginseng. Changes in root size and active component accumulation of P. notoginseng are influenced by related genes and affected by changes in the cultivation environment. Increased expression of PnGAP and PnEXPA4 results in the thicker cell wall and a higher content of lignin, cellulose, and callose in P. notoginseng of cultivation era. Higher expression of GGPPS, FPS, CYP716A47, and CYP716A53v2 contributes to a higher content of ginsenosides, while lower expression of SAT might result in a lower content of dencichine. A PnPHL8 transcriptional factor participates in transcriptional regulation of PnGAP, PnCYP716A47, and PnGGPPS3, while dotted line between PnEXPA4 and PnGGPPS3 indicates that a probable positive correlation may exist.