Morphological changes during P. notoginseng taproot thickening
We measured the dynamic growth indexes of the taproots of one -year-old P. notoginseng plants monthly. The results showed that the seed root germinated in March, while thickening took place from May to July and stabilized from August to November (Fig. 1a). However, the taproot elongation was not significant until July in comparison with March (Fig. 1b). Both the fresh and dry weight of the taproot increased with the growth period, but there was no significant difference from August to November (Fig. 1c). Similarly, the total saponins also increased with the growth month, but there was no significant difference from September to November (Fig. 1d). This suggested that fresh weight is indicative of saponin content during the first year of P.notoginseng growth.
Taproot thickening is associated with secondary meristem activity. We observed the anatomical structure of the taproot during different months of growth. The results showed that the vascular bundles were not well developed in the seed root stage in March (Fig. 2a). The division of the secondary cambium initiated taproot thickening in May (Fig. 2b, c) and divided continuously in July, which produced secondary phloem cells outward and secondary xylem cells inward that promoted rapid thickening of the taproot (Fig. 2d, e). From October, the thickening rate of the taproot slowed down and tended to stabilize (Fig. 2f, g). These results allowed us to identify four key stages associated with taproot thickening in P. notoginseng: seed root stage (March), initial thickening stage (May), rapid thickening stage (July) and stable thickening stage (November).
RNA sequencing analysis of the developing taproot of P.notoginseng
To explore the molecular basis of the morphological changes during taproot thickening, RNA sequencing (RNA-Seq) analysis of the taproots at four different stages was performed. Principal component analysis (PCA) revealed that the 12 samples could be clearly assigned to four groups. There was a significant difference between March and May, while July and November clustered together, suggesting that the overall transcriptomic profiles between the seed root and initial thickening stages were distinct, while those of the rapid and stable thickening stages were similar (Fig. 3a). In order to obtain reliable gene expression profiles, reads with log2 |Fold Change| > 1 and RPKM values >10 were selected to annotate the differentially expressed genes (DEGs). A total of 9079 up-regulated DEGs and 20,878 down-regulated DEGs were derived from the comparison between the different stages. The number of down-regulated DEGs was higher than that of the up-regulated genes in March, May and July, whereas the opposite occurred in November (Fig. 3b). The up- or down-regulated DEGs in the different taproot thickening stages are listed in Additional file 1: Table S1. A Venn diagram of the DEGs showed that none were up-regulated consistently in May, July and November vs. March, while 12 genes were up-regulated consistently in May and July vs. March (Fig. 3c). By contrast, four genes were down-regulated consistently in May, July, and November vs. March, while 43 genes were down-regulated in May and July vs. March (Fig. 3d). As initial and rapid thickening are the key stages for taproot development, the consistently up-regulated or down-regulated genes in May and July vs. March play an important role in taproot development in P. notoginseng. In fact, the taproot almost stopped thickening in September (Fig. 1a). Therefore, the change in gene expression profile was not significant in the stable thickening stage, as reflected by the number of DEGs in November vs. July (Fig. 3b). The high-quality reads obtained in this study were deposited in the NCBI SRA database (accession number: SUB5611240).
The DEGs were further categorized and characterized according to the functional category defined by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. GO analysis showed that the DEGs were mainly involved in “metabolic process,” followed by “cell process” and “single-organism process” (Additional file 2: Figure S1). Metabolic processes analysis by KEGG showed that the DEGs were predominantly enriched in “plant hormone signal transduction pathway,” “starch and sucrose metabolism pathway,” and “phenylpropanoid biosynthesis pathway” (Fig. 4), suggesting that these pathways may be closely related to taproot thickening in P. notoginseng.
Identifying critical genes controlling taproot development in P. notoginseng
According to the Venn diagram, 12 DEGs were significantly up-regulated in the pairwise comparisons among May vs. March, July vs. March, and July vs. May. These genes were mainly related to “carbohydrate and storage metabolism,” including genes encoding RNase-like major storage protein, Beta-amylase (BAMY) 5, Alpha-1,4 glucan phosphorylase L isozyme, Starch branching enzyme (SBE) I and Vacuolar protein sorting-associated protein 32 homolog 2, and “signal transduction and transcription,” including genes encoding Protein phosphatase 2C 37-like, Two-component response regulator-like APRR7, and DA1-related protein 5 (Table 1). In contrast, 43 down-regulated DEGs overlapped among the above-mentioned comparisons and were mainly involved in “signal transduction and transcription,” “stress/defense response,” and “other metabolisms,” including lipid metabolism, secondary metabolism, and cell wall metabolism (Table 2). Noticeably, four genes showing high homology with Delta(12)-fatty-acid desaturase FAD2-like (AtAT3G12120) were identified as consistently down-regulated during taproot thickening (Table 2). These results suggested that the genes associated with signal transduction, transcriptional regulation and metabolic pathways play diverse roles in taproot thickening in P. notoginseng.
To validate the reliability of the transcriptomic data, we selected 16 DEGs that include 12 DEGs mentioned above for the quantitative real-time PCR (qRT-PCR) analysis. There was a good correlation (r = 0.83) between the transcriptomic data and the qRT-PCR results (Additional file 3: Figure S2). These results indicated that the transcriptomic data could reflect the transcriptional changes during the thickening process in the taproots of P. notoginseng.
Differential expression of TFs
A total of 237 DEGs were identified and assigned to 30 TF families (Additional file 4: Table S2). Among them, 97 DEGs encoding APETALA2/ethylene-responsive factor (AP2/ERF), WRKY, bHLH, NAC domain containing protein (NAC), BRI1-EMS-SUPPRESSOR 1 (BES1), Cys2-His2 zinc finger protein (C2H2), and Auxin response factor (ARF), have previously been implicated in plant growth and development [22–28] (Fig. 5). APETALA2/ethylene response factor (AP2/ERF) TF is one of the largest superfamilies in the plant kingdom, indicating that different members play a specific role in different taproot thickening stages (Fig. 5). Genes encoding the C2H2 and ARF families were predominantly up-regulated in March, while genes encoding the WRKY, bHLH, NAC, and BES1 families were up-regulated mainly in March and May (Fig. 5). These results suggest that the up-regulated TF families may be involved in the early developmental regulation of P.notoginseng taproot thickening.
Differential expression of hormone signaling transcripts
The involvement of plant hormones, including indole-3-acetic acid (IAA), cytokinin (CTK), gibberellin (GA), ethylene (ETH), jasmonate (JA), and brassinosteroid (BR), in the development and formation of the roots has been investigated previously [29, 30]. Our KEGG analysis showed that “plant hormone signal transduction pathway” was predominantly enriched during taproot thickening in P. notoginseng (Fig. 4), with 93 DEGs grouped into the IAA, BR, abscisic acid (ABA), GA, ETH, JA, zeatin (ZT), salicylic acid (SA), and CTK pathways. While DEGs associated with the GA signaling pathway were up-regulated mainly in November, DEGs related to the ABA signaling pathway were significantly up-regulated in March and November (Fig. 6). To verify whether the gene expression was correlated with hormone metabolism, the endogenous IAA and JA contents at four taproot development stages were measured using liquid chromatography-mass spectrometry (LC-MS). The results showed that IAA and JA accumulated predominantly in March and May and both decreased significantly with the gradual thickening of the taproot, which is consistent with the transcriptome data (Additional file 5: Figure S3; Fig. 6). These results indicated that different hormones may have synergistic or/and antagonistic functions in the regulation of P. notoginseng taproot thickening.
Differential expression of genes related to starch and sucrose metabolism
The enrichment of DEGs related to “starch and sucrose metabolism” suggests their important role in P. notoginseng taproot thickening (Fig. 4). Metabolic pathway analysis showed that glucose, fructose, sucrose, and starch could be metabolically connected (Fig. 7a). Here, 61 DEGs encoding enzymes related to starch and sucrose metabolism were identified as up-regulated mostly in May, July, and November, including alpha-amylase genes (AMY), BAMY, starch synthase genes (SS), and granule-bound starch synthase genes (GBSS) involved in starch metabolism, and invertase genes (INV), sucrose synthase genes (SuS), and sucrose-phosphate synthase genes (SPS) involved in sucrose metabolism (Fig. 7b). To further clarify whether taproot thickening in P. notoginseng is directly associated with changes in carbohydrates, the primary metabolites at four taproot development stages were assessed by absolute quantitative analysis using Nuclear Magnetic Resonance (NMR) spectroscopy. PCA revealed that the four samples could be distinctly separated (Additional file 6: Figure S4). Samples collected from July and November clustered closely, suggesting that the overall metabolite profiles were similar between the rapid and stable thickening stages. The differential abundance profiles indicated that five metabolites, including fructose, glucose, sucrose, arginine and malate, differed significantly during taproot thickening (Fig. 8a). Among them, fructose and glucose accumulated dominantly in March and May, while sucrose accumulated mostly in May, July, and November. This was basically consistent with the transcriptome data (Fig.7b; Fig. 8a). It is presumed that sucrose is degraded into fructose and glucose for conversion into other organic matters during the early stage, while taproot thickening is also accompanied by increased malate and arginine accumulation (Fig. 8b).
Differential expression of cell wall metabolism transcripts
The cell wall not only strengthens the plant body, but also has key roles in plant growth, cell differentiation, cell expansion, intercellular communication, water movement and defense [31, 32]. In the present study, a total of 96 DEGs encoding enzymes that are involved in cell wall modification, synthesis, and degradation were identified. Among them, the largest number of genes was up-regulated in May (Fig. 9), suggesting that the changes in cell wall components are necessary for the initiation and induction of taproot thickening in the early stages in P. notoginseng. In particular, DEGs encoding extension (EXT), glycosyltransferases (GT), and pectinesterase (PE) were mostly up-regulated in March and May, while those encoding expansin (EXP) were highly expressed in May and July (Fig. 9).