## 1 Introduction
### Cis-Acting Elements Revealed the Possible Transcription Regulation of LerHMGRs in L. erythrorhizion  
The PlantCARE tool was used to scan the 2000 bp upstream promoter regions of _LerHMGR_ genes for putative cis-acting elements that regulate their expression, and a total of 2019 cis-acting elements of 80 types were found in all _LerHMGRs_ promoters (Table S5). Among them, 17 types of light-responsive elements were identified, including Box 4, the GA-motif, the G-box, and the MYB binding site involved in light responsiveness (MRE), which are consistent with the photoinhibition regulation of shikonin biosynthesis [43] (Figure 3). Meanwhile, 12 hormone-related cis-elements were identified, with the majority of _LerHMGR_ promoters containing responsive binding sites--MeJA-responsive element (CGTCA-motif, TGACG-motif), ethylene-responsive element (ERE), and auxin-responsive element (TGA-element), implying that _LerHMGR_ genes may be regulated by jasmonic acid, ethylene, or auxin, all of which have been shown to promote shikonin biosynthesis [44; 45; 46] (Figure 3). There are 15 distinct types of stress-related elements, including the anaerobic induction regulatory element (ARE), defense- and stress-responsive element (TC-rich repeats), low-temperature responsiveness element (LTR), and the MYB binding site involved in drought-inducibility (MBS), and these stress conditions are analogous to those found in shikonin-producing species (Figure 3). Additionally, the presence of numerous growth-related components suggests that _LerHMGRs_ may contribute to the growth of _L. erythrorhizion_ (Figure 3). Overall, certain elements such as Box 4, STRE, ARE, TGACG-Box, and ERE were identified as high-frequency elements in the promoters of _LerHMGRs_ (Figure S3). Cluster analysis revealed that _LerHMGR2_ contained the fewest light-responsive elements and total cis-acting elements (Figure 3). To summarize, these results suggest that transcript levels of _LerHMGRs_ may be regulated by components in light, hormone, and stress response regulatory pathways associated with the accumulation of shikonin in _L. erythrorhizion_. Expression Patterns Revealed the Possible Critical Role of LerHMGR1 and LerHMGR2 for the Biosynthesis of Shikonin and Its Derivatives  
Given that the production of secondary metabolites is associated with the tissue-specific expression of related genes [47], and that shikonin accumulates abundantly in the root [48] and M9 in the dark [49], genes involved in the shikonin pathway should be highly expressed under these conditions. We first screened for major _LerHMGRs_ involved in shikonin biosynthesis using publicly available transcriptome data from six tissues (the mature root (MR), periderm of mature root (PD), cortex of mature root (CT), stele of mature root (SE), leaves + stems (ML), and flowers (FL)) of the _L. erythrorhizion_ seedlings and two growth conditions (wild-type hairy root tissue cultures grown in M9 in the dark versus B5 in the light) [22; 50]. The results showed most _LerHMGRs_ had expression levels in the mature root, root periderm, and hairy root growing in M9 under darkness, which is the primary site for the accumulation of shikonin and its derivatives, but _LerHMGR2_ had the highest level of expression, followed by _LerHMGR1_ (Figure 4A). The TPM value of _LerHMGR2_ in the mature root and periderm was 19-3880 times and 8.4-20,058 times higher than that of other genes except _LerHMGR1_, respectively (Figure 4A). Additionally, the TPM value of _LerHMGR1_ in the mature root was 4.43-902.37 times that of other genes, and that of _LerHMGR1_ in periderm was 2.77-6626 times that of other genes (Figure 4A). Furthermore, the TPM values of _LerHMGR2_ and _LerHMGR1_ in hairy roots under M9 dark culture were 17.18-491.2 times and 5.88-190.34 times those of other genes, respectively (Figure 4A).