Korean ginseng (P. ginseng) has long been used as one of the most important medicinal root herbs for the treatment of various diseases and as a health supplement in the oriental countries (Hu 1976; Mahady et al. 2000). Korean ginseng’s perennial growth and the gradual accumulation of medicinal components in its roots resulted in the development of a special cultivation method in Korea, which was cultivated in same area for over six years. In other words, the yield of the Korean ginseng grown over time is closely related to the secondary development of storage roots. Recent developments in sequencing technology have resulted in an improvement in the accuracy of the information regarding the ginseng genome, and it has been discovered that multiple genes play critical roles in the process of growth and development (Hong et al. 2018; Hong et al. 2021a; Jayakodi et al. 2018; Kim et al. 2018). In particular, a number of genes that are associated to the secondary growth of ginseng roots and the manufacture of saponin have been found (Luo et al. 2011; Xu et al. 2017).

Homeostatic maintenance of meristem cells in the cambium are crucially regulated by plant hormones and their crosstalk during plant secondary growth (Fischer et al. 2019; Hoang et al. 2020). Particularly, local auxin accumulation in the cambium and xylem precursor cells and sequential activation of downstream signaling pathways in these cells are necessary for vascular patterning and secondary development (Jang et al. 2018; Smetana et al. 2019). Many studies have demonstrated that auxin maxima redistribution via PAT (Polar Auxin Transport) is critical for plant growth and development (Adamowski and Friml 2015; Brackmann et al. 2018; Omelyanchuk et al. 2016; Ruonala et al. 2017). WUSCHEL-related HOMEOBOX4 and other xylem-related genes are regulated by auxin accumulation and activation of downstream signalling, which in turn affects cambial cell activity and xylem differentiation (Fischer et al. 2019). Consistently, it was reported that auxin accumulation rose progressively from the active cambial zone to the xylem initiation zone during the secondary growth, but it fell drastically in mature xylem cells during wood formation (Kucukoglu et al. 2017). These findings suggest that appropriate vascular patterning and radial expansion during post-embryonic plant development are likely dependent on local control of auxin homeostasis and accurate maximum formation in the vascular cambium.

Storage roots of plants, like shoot radial secondary growth, are generated as a result of the thickening of primary or adventitious roots. The vascular cambium is primarily responsible for this root thickening process, which is known as the root secondary growth (Hoang et al. 2020). The vascular cambium is formed by the division and reorganization of cells originating from the procambium and its neighboring pericycle cells in the root’s procambium. These cells then give rise to secondary xylem and phloem cells, which are actively dividing and differentiating into storage parenchyma cells (Fischer et al. 2019; Hoang et al. 2020). As a result, the radial growth regulated by the vascular cambium determines the growth rate and yields of the most of root crops.

Walls Are Thin 1 (WAT1), a plant-specific tonoplast localized plant-drug/metabolite exporter (P-DME) family, plays critical roles in the regulation of local auxin homeostasis in xylem precursor cells during plant secondary growth (Lee et al. 2021; Ranocha et al. 2010; Ranocha et al. 2013). Brassinosteroid-mediated upregulation of WAT1 regulates auxin accumulation spatiotemporally, which were critical for secondary xylem and wood formation in plants (Lee et al. 2021). Although the positive role of WAT1 in shoot secondary growth has been investigated, little is known regarding P. ginseng’s storage root thickening function. In this study, we identified four PgWAT1-like genes (PgWAT1s and PgWAT2s) whose expression was significantly enriched in P. ginseng with highly developed storage roots by nitrate treatment (Geem et al. 2022). A functional analysis with a tomato wat1 loss-of-function mutant indicated that PgWAT1-1 and PgWAT2-1 could stimulate plant secondary growth by genetically complementing auxin transporter activity.

Phylogenetic analysis and protein sequence alignments

The protein sequences of PgWAT1-related genes including PgWAT1-1 (Pg S2763.12), PgWAT1-2 (Pg S2027.39), PgWAT2-1 (Pg S4250.12), and PgWAT2-2 (Pg S7019.3) were selected. A phylogenetic tree based on protein sequence alignment was generated using MEGA version 7.0 software by the neighbor-joining method with a bootstrap value of 1000 (Hall and Horowitz 2013; Kumar et al. 2016). An online program, SMS (http://www.bioinformatics.org), was applied to align the protein sequence. Percentage of sequences that must agree for identity or similarity coloring to be added was 70%.

Protoplast transient expression assay

The full-length cDNAs of PgWAT1-1, PgWAT2-1, SlWAT1 and AtVAMP were cloned into plant expression vectors containing GFP or mCherry sequence tags in the C terminus driven by the 35S::C4PPDK promoter as previously described (Lee et al. 2021). For protoplasts transient expression assays, about 4 × 10⁴ protoplasts were transfected with 20 µg of plasmid DNA and then incubated under constant light condition at 23°C for 6 h. GFP and mCherry were observed with a fluorescence microscope (Olympus BX53).

Transgenic plants and immunoblotting assay

Solanum lycopersicum L. Slwat1-copi mutant (Lee et al. 2021), was used as the controls and as the genetic backgrounds for all transgenic lines. The tomato seeds were surface-sterilized in 70% ethanol and 50% bleach, and then plated on MS medium (Duchefa, Haarlem, Netherlands), 1% sucrose and 0.8% phytoagar under an environmentally controlled growth room (25°C, 16-h light/8-h dark). 10 day-old seedlings were transplanted to the mixture of peat-vermiculite (50:50 v:v) and grown in the green house (22°C, 16-h light/8-h dark). Transgenic tomato plants were generated as described previously (Lee et al. 2019). cDNA fragments of PgWAT1-1 and PgWAT2-1 was cloned into Pbi121 containing the 35S promoter and FLAG tag sequences as described previously (Lee et al. 2021). All transgenic lines were generated by Agrobacterium-mediated methods. The transgene expression was confirmed by immunoblot analysis that quantified FLAG gene expression in the T2 generation. Total proteins from seedlings were extracted with protein extraction buffer (50 mM Tris-HCl (pH 7.5), 75 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 1× protease inhibitor cocktail (Roche), and 1% Triton X-100). Total protein (3-20 µg) was separated by SDS-PAGE (10% polyacrylamide), transferred to a polyvinylidene difluoride membrane and immunodetected using 1/2,000 dilution of a peroxidase-conjugated anti-FLAG antibody (Sigma).

Histological sections and microscopy

Fresh hand-cut cross sections of internode 2 from tomato plant stems were prepared at 45 DAG. For paraffin sectioning, tissues were dehydrated, embedded in paraffin, sliced into 10 μm-thick sections and mounted onto slides. After dewaxing with Histo-Clear, the slides were dehydrated and stained with 1% safranin and 0.5% fast-green. The sections were mounted in Permount (Thermofisher) and were imaged with a Nikon Ti-U Series. The autofluorescence signals of secondary xylem cell walls were observed and photographed at an excitation wavelength of 355 nm (Donaldson and Radotic 2013) with a fluorescence microscope (Nikon). The number of SCW-deposited xylem cell rows was counted on a straight line traced from the last procambium cell layer to the inner xylem cells facing the center of the stem as described previously (Lee et al. 2019).

One of the key elements that influences the yield and quality of P. ginseng is the promotion of secondary growth of the storage roots. Recent research has demonstrated that treatments with exogenous nitrate or gibberellins (GAs) significantly accelerated the storage root secondary growth of P. ginseng (Geem et al. 2022; Hong et al. 2021b). In these studies, the protein-protein interaction network analysis of two secondary growth promoting factors revealed that the auxin activated signaling pathway would be fundamentally integrated into the hormonal regulation networks for storage root formation (Geem et al. 2022; Hong et al. 2021b). To further investigate the core genetic networks affecting the auxin-connected storage root thickening process in P. ginseng, we reanalyzed the RNA-seq data of nitrate-treated roots of 1-year-old P.ginseng (Geem et al. 2022). The functional categories of an auxin efflux-related term were significantly enriched by the nitrate treatment, according to gene set enrichment analysis (GSEA) of differentially expressed genes (DEGs) (FDR: 0.0017; Fig. 1A). The formation of auxin maxima in cambium stem cells through spatiotemporal regulation of auxin transporters is essential for plant radial secondary growth (Fischer et al. 2019; Smetana et al. 2019). Key auxin transporters, such as PINs, WAT1s, and ABCGs, were found to be differentially regulated in the nitrate-treated ginseng roots according to the analysis of expression patterns of a leading-edge group for the creation of enrichment scores in the GSEA (Fig. 1B). The exogenous nitrate treatment increased the expression of many WAT1-like genes, suggesting that the WAT1-mediated local auxin accumulation in the vascular cambium might play a key role in ginseng storage root thickening.

Figure 1. Auxin efflux-related genes were significantly enriched in the nitrate-promoted root growth of P. ginseng. The enrichment plot for the auxin efflux term (A) and an expression heatmap of leading-edge genes (B) of this pathway (FDR = 0.017) were obtained from the RNA-seq data of the roots of nitrate-treated P. ginseng (Geem et al. 2022)

We then explored the evolutionary conservation of the upregulated PgWAT1-like genes in the nitrate-treated ginseng roots as a tonoplast-anchored auxin efflux carrier functioned in facilitating the secondary plant growth (Lee et al. 2021; Ranocha et al. 2013). Through phylogenetic analysis, the relationship between the protein sequences of the WAT1 family of tomato and Arabidopsis was discovered (Fig. 2A). Phylogenetic analysis showed that two clades of PgWAT1-like genes were related to AtWAT1 and SlWAT1 (Fig. 2A). We named PgWAT1-1, PgWAT1-2, PgWAT2-1, and PgWAT2-2. We identified the primary EamA-like transporter domains present in the WAT1 proteins of Arabidopsis, tomato, and P. ginseng using comparative protein sequence analysis (Fig. 2B). The anticipated protein topology of AtWAT1, SlWAT1, PgWAT1-1, and PgWAT2-1 was then determined. As shown in Fig. 2C, all of the examined WAT1 proteins contain 10 expected transmembrane helix sequences based on the calculation of G values across the amino acid sequences. These imply that PgWAT1-1 and PgWAT2-1 are likely membrane proteins with 10 transmembrane domains (Fig. 2D).

Figure 2. Identification of WAT1-like genes in the P. ginseng genome. (A) Phylogenetic analysis of AtWAT1s, SlWAT1s, PgWAT1-1 (GenBank accession number OP115969), PgWAT1-2 (OP115970), PgWAT2-1 (OP115971), and PgWAT2-2 (OP115972). The phylogenetic tree was constructed using the MEGA7 program. Bootstrap values were obtained by 1000 bootstrap replicates. At, Arabidopsis thaliana; Sl and Solyc, Solanum lycopersicum; Pg, Panax ginseng. (B) Amino acid sequence alignment of the WAT1 proteins of A. thaliana, tomato, and P. ginseng constructed using SMS (https://www.bioinformatics.org). Conserved domains were predicted using the Pfam v33.1 database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). (C) Protein topology of AtWAT1, SlWAT1, PgWAT1-1, and PgWAT2-1 predicted by TOPCONS (https://topcons.net/pred/) and their calculated ΔG values across the sequence. (D) Putative diagrams of tonoplast-anchored PgWAT1-1 and PgWAT2-1 proteins. Red and blue lines indicate intra- and extra-cellular domains, respectively. TM, transmembrane domain

WAT1 is a tonoplast-localized auxin efflux carrier protein that regulates local auxin homeostasis in xylem precursor cells during radial secondary growth (Lee et al. 2021). The subcellular localization of PgWAT1-1 and PgWAT2-1 proteins was next examined to determine whether PgWATs has a biological function in the vacuole as similar to our previous findings (Lee et al. 2021). In Arabidopsis protoplasts, the GFP-tagged PgWAT1-1 and PgWAT2-1 colocalized with the tonoplast marker AtVAMP-RFP (Geldner et al. 2009), which was identical to the tonoplast-localized SlWAT1-GFP (Fig. 3).

Figure 3. Subcellular localization analysis of PgWAT1-1 and PgWAT2-2 proteins in Arabidopsis protoplasts. SlWAT1, PgWAT1-1, and PgWAT2-1 full-length coding sequences were fused to the GFP reporter gene. The tonoplast was visualized using the AtVAMP-RFP tonoplast marker. GFP and RFP fluorescence images were merged. Scale bar = 50 µm

To further investigate the physiological roles of PgWAT1s in plants, we examined whether overexpressing them restored the WAT1-defected secondary growth phenotypes in a slwat1 loss-of-function mutant, slwat1-copi tomato plants (Lee et al. 2021, Fig. 4). We generated two independent PgWAT1-1 and PgWAT2-1 overexpressing tomato plants in a slwat1-copi mutant background. The stem diameter of slwat1-copi tomato mutants was significantly increased as a result of ectopic expression of PgWAT1-1 and PgWAT2-1 (Fig. 4A, 4B). The expression levels of the PgWAT1-1 and PgWAT2-1 proteins in the transgenic slwat1-copi tomato plants were further confirmed with a western blot analysis (Fig. 4C). Secondary growth defects in the slwat1-copi tomato mutant were caused by a slower rate of secondary xylem differentiation. We next examined histological sections to evaluate whether PgWAT1-1 and PgWAT2-1 overexpression facilitated in the development of secondary xylem in tomato stems. Consistent with the increased stem diameter phenotype, the PgWAT1-1 and PgWAT2-1 overexpressing slwat1-copi tomato plants exhibited an increase in secondary xylem formation (Fig. 4D, 4E). Taken together, these results indicate that the PgWAT1-1 and PgWAT2-1 are enough to restore the physiological function of SlWAT1 in plant secondary growth.

Figure 4. PgWAT1-1 and PgWAT2-1 function in facilitating secondary growth in plants. (A) Representative shoot growth phenotypes of Slwat1 loss-of-function mutant (Slwat1-copi) and Slwat1-copi plants overexpressing PgWAT1-like genes (PgWAT1-1/Slwat1-copi and PgWAT2-1/Slwat1-copi) at 40 days after germination (DAG). Scale bar = 5 cm. (B) Quantification of secondary growth (diameter of the 2^nd internode) of the indicated plants at 45 DAG. Dots represent all values [n = 6; P < 0.05; repeated measures analysis of variance (ANOVA) with Tukey’s multiple range test]. (C) Western blot image of PgWAT1-1 and PgWAT2-1 protein levels in Slwat1-copi plants. (D) Representative images of secondary xylem in the stems of the tomato genotypes (Fig. 4B). Autofluorescence was used to visualize secondary cell walls (SCW) in xylem tissue, and secondary xylems were colored in bright field images. Scale bar = 200 µm. (E) Quantification of xylem cell numbers in the indicated genotypes. Dots represent all values. Each box is located between the upper and lower quartiles, and the whiskers represent the lowest or highest data point within the 1.5 interquartile range of the lower or upper quartile. The mean is represented in the boxes by thick horizontal lines, and all values are represented by dots [n = 20; P < 0.05; repeated measures ANOVA with Tukey’s multiple range test]

In this study, we discovered WAT1-like genes in P. ginseng genome, which are known to promote plant secondary growth. In order to facilitate the formation of storage roots and the accumulation of different medicinal compounds, ginseng must be grown for at least 6 years. The majority of research on plant growth and development has been done using an annual herbaceous model plant system, like Arabidopsis, tomato and rice. However, in light of the recent reported research on GA signaling and nitrate assimilation in the development of ginseng’s storage roots (Geem et al. 2022; Hong et al. 2021b), our study will provide critical ideas for developing strategies to support the perennial root crop P. ginseng’s secondary growth. Particularly, the physiological significance of auxins in the secondary root growth of P. ginseng, as well as their redistribution, are considered to require more detailed further study in the future. The knowledge of P. ginseng’s WAT1 genes obtained through this study will play a significant role in the advancement of biotechnology for breeding and expanding the production of P. ginseng.

This work was supported by the the Research Program 2021 of the Korean Society of Ginseng and conducted during the research year of Chungbuk National University in 2022