The effects of different hormone combinations on the growth of Panax notoginseng anther callus based on metabolome analysis

Abstract

Panax notoginseng saponins (PNS), the primary active components of Panax notoginseng (Burk.) F.H.Chen, a traditional and precious Chinese medicinal herb, are mainly derived from the roots of the plant. However, due to the long cultivation period and specific environmental requirements, the PNS supply is often limited. And, callus cultures of P. notoginseng, which grow rapidly, have short production cycles, and can be cultured under controlled conditions, provide a more efficient source for the quick acquisition of saponins. In this study, anthers of P. notoginseng were used as explants, and twelve hormone combinations were tested to induce callus formation. Eight kinds of hormone combinations successfully induced P. notoginseng anther callus. Among these, callus induced by combinations 5 and 7 had the highest saponin content, while those induced by combinations 1 and 3 exhibited the highest relative growth rates. Metabolomic analysis of these four callus types revealed that there were a total of 99 differential metabolites between combinations 5 and 7, 30 between combinations 1 and 3, 123 between combinations 3 and 7, and 116 between combinations 1 and 5. Further analysis showed that the tricarboxylic acid (TCA) cycle metabolites in callus induced by combinations 1 and 3 were significantly upregulated, with corresponding genes showing high expression levels, increased ATP accumulation, and low responses of the auxin response factor PnARF-3 and cytokinin response factor PnCRF-3. The abundance of metabolites in the PNS biosynthesis pathway in callus induced by combinations 5 and 7 increased significantly, with related genes showing high expression levels, increased IPP accumulation, and high responses of PnARF-3 and PnCRF-3. Overexpression of PnARF-3 and PnCRF-3 in callus induced by combination 3 promoted the production of IPP and saponins while reducing ATP production. In conclusion, different hormone combinations affect the distribution of Acetyl-CoA through PnARF-3 and PnCRF-3, resulting in the relative growth rate and saponin of P. notoginseng anther callus differences.

Keywords: P. notoginseng callus; TCA cycle; auxin response factor; cytokinin response factor; saponin synthesis; secondary metabolite.

Figure 1

Flow chart of PNS synthesis, with Acetyl-CoA as the starting material. The precursor isopentenyl pyrophosphate (IPP) was synthesized by 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MVK) and phosphomevalonate kinase (PVK). IPP was via geranyl pyrophosphate synthase (GPS), farnesyl diphosphate synthase (FPS), squalene synthase (SS) and squalene epoxidase (SE) synthetic skeleton 2,3-oxidosqualene. 2,3-oxidosqualene is synthesized PNS by Dammarenediol-II synthase (DS), CYP450 and UGT. The blue boxes denote the substances involved in the synthesis process, while the red boxes indicate the key genes.

Figure 2

The appearance and structure of P. notoginseng flower, bud and anther. (A) P. notoginseng Flower; (B) Bud, within the red circle is the anther; (C) The scanning electron microscope result of anther was columnar.

Figure 3

Morphological structure of P. notoginseng anther callus with different hormone combinations. (A–D) Appearance structure of P. notoginseng anther callus with hormone combinations No.1-No.4; (E) Observation of cytological structure of non-embryogenic callus by microscope (No.3); (F–I) Appearance structure of P. notoginseng anther callus with hormone combinations No.5-No.8; (J) Observation of cytological structure of embryogenic callus by microscope (No.7).

Figure 4

Illustrates the callus induction rate and relative growth rate of P. notoginseng anther with different hormone combinations. (A) Callus induction rate of P. notoginseng anther in hormone combination No.1-No.8; (B) Callus relative growth rate of P. notoginseng anther in hormone combination No.1-No.8. Data are expressed as average values ± SE (n=3), different letters indicate significant differences at p < 0.05 (Student’s test).

Figure 5

Displays the saponin content in Callus of P. notoginseng anther with different hormone combinations. (A) HPLC chromatogram of P. notoginseng anther callus in hormone combination No.1-No.8; (B) The content of total saponins of seven monomers in P. notoginseng anther callus in hormone combination No.1-No.8 was determined by HPLC. Different letters indicate significant differences at p < 0.05 (Student’s test).

Figure 6

Analysis the metabolite of P. notoginseng anther callus with different hormone combinations; (A) The proportion of different metabolites in the anther callus of P. notoginseng; (B) PCA principal component analysis diagram of different hormone combinations; (C) The number of different metabolites in different comparison groups; (D) Venn diagram; (E) The cluster heat map of the 20 metabolites of the most up-regulated and down-regulated in four hormone combinations.

Figure 7

Illustrates the TCA cycle, MVA Pathway and downstream of saponins synthesis metabolic pathway. Acetyl-CoA serves as a precursor, part of it enters the TCA cycle to synthesize citrate acid, cis-aconitate acid, isocitrate, fumarate acid, malate acid and other substances to produce ATP. Some of these substances are directed into the MVA pathway to generate mevalonate, IPP and 2,3-Oxidosqualene. 2,3-Oxidosqualene entered the saponin synthesis pathway, and further synthesized ginsenoside Rg1, ginsenoside Rf, ginsenoside Rg3, ginsenoside Rd and ginsenoside Rb1. The red box highlights precursor substances, the yellow box denotes TCA cycle, the blue box represents MVA pathway, and the green box indicates downstream pathway of saponin synthesis. The red font identifies the detected metabolites in the callus of P. notoginseng anther, while the black font denotes the undetected metabolites in the callus of P. notoginseng anther.

Figure 8

Presents the contents of ATP and IPP in callus of P. notoginseng anther with different hormone combinations. (A) Determination of ATP content of P. notoginseng anther callus in different hormone combinations by kit; (B) Determination of IPP content of P. notoginseng anther callus in different hormone combinations by kit. Data are expressed as average values ± SE (n=3), different letters indicate significant differences at p < 0.05 (Student’s test).

Figure 9

Shows the expression levels of various genes in the of P. notoginseng anther callus, as determined by RT-qPCR. The figure includes TCA cycle related genes PnCSE-1 (citrate synthase 1), PnCSE-2 (citrate synthase 2), PnCSE-3 (citrate synthase 3), PnIDE-1 (isocitrate dehydrogenase 1), PnIDE-2 (isocitrate dehydrogenase 1), PnMDE (malate dehydrogenase), MVA pathway related genes PnHMGR-1, PnHMGR-2, PnHMGR-3, PnMVK and PnPVK, saponin biosynthesis pathway related genes PnFPS, PnGPS, PnSS, PnSE, PnDS, PnCAS, auxin response factor related genes PnARF-1 (Auxin response factor 1), PnARF-2 (Auxin response factor 2), PnARF-3 (Auxin response factor 3), PnARF-4 (Auxin response factor 4), and cytokinin response factor related genes PnCRF-1 (Cytokinin response factor 1), PnCRF-2 (Cytokinin response factor 2), PnCRF-3 (Cytokinin response factor 3), PnCRF-4 (Cytokinin response factor 4). Red indicates high gene expression, while blue indicates low gene expression.

Figure 10

Shows various analyses of P. notoginseng anther callus following the overexpression of PnARF-3 and PnCRF-3. (A) The expression levels of PnARF-3 and PnCRF-3 in callus of P. notoginseng anther after transformation were detected by RT-qPCR; (B) ATP content in callus of P. notoginseng anther after overexpression of PnARF-3 and PnCRF-3 was detected by kit; (C) IPP content in callus of P. notoginseng anther after overexpression of PnARF-3 and PnCRF-3 was detected by kit; (D) Total saponins content in callus of P. notoginseng anther after overexpression of PnARF-3 and PnCRF-3 was measured by kit; (E) The contents of total saponins of seven monomers in callus of P. notoginseng anther after overexpression of PnARF-3 and PnCRF-3 were detected by HPLC. Data are expressed as average values ± SE (n=3), different letters indicate significant differences at p < 0.05 (Student’s test).

Figure 11

Shows the expression levels of various genes in the callus of P. notoginseng anther following the overexpression of PnARF-3 and PnCRF-3, as determined by RT-qPCR. The genes are categorized as follows: TCA cycle related genes PnCSE-1, PnCSE-2, PnCSE-3, PnIDE-1, PnIDE-2, PnMDE, MVA pathway related genes PnHMGR-1, PnHMGR-2, PnHMGR-3, PnMVK, PnPVK, saponin biosynthesis pathway related genes PnFPS, PnGPS, PnSS, PnSE, PnDS and PnCAS. Red indicates high gene expression, while blue indicates low gene expression.

Figure 12

Illustrates different hormone combinations affect TCA cycle, MVA pathway and saponin synthesis pathway in P. notoginseng anther callus through PnARF-3 and PnCRF-3. Different hormone combinations affect the expression levels of PnARF-3 and PnCRF-3 in the callus, and overexpression of PnARF-3 and PnCRF-3 leads to a reduction in the entry of Acetyl-CoA into the TCA cycle. This results in decreased ATP synthesis and a slower growth rate. Concurrently, more Acetyl-CoA is directed into the MVA pathway, which promotes the synthesis of IPP and enhances saponin accumulation. In the figure, the blue box represents the TCA cycle, while the red box highlights the MVA pathway and saponin synthesis pathway.