## 3 Discussion
For example, _LerHMGR6_ is mainly highly expressed in flowers, which may be related to the synthesis of volatile terpenoids or pigment lipid-soluble terpenoids in flowers [58]. _LerHMGR7_, on the other hand, is highly expressed in leaves and may be involved in the accumulation of terpenoids. Similar to the expression levels of the _TmHMGR_ gene in _Taxus media_ which was generally consistent among leaves, roots, and stems, _LerHMGR10_ may also be constitutionally expressed and involved in regulating basic physiological metabolism [59]. _LerHMGR1_ and _LerHMGR2_ are the only members of the _LerHMGR_ gene family that are highly specifically expressed in both the root and M9 dark culture, and their catalytic functions of MVA biosynthesis have been demonstrated by enzyme activity experiments in vitro. Regrettably, we did not clarify the contribution ratio of LerHMGR1 and LerHMGR2 to the accumulation of shikonin. This issue needs to be explored further by comparing the kinetic properties of LerHMGR1 and LerHMGR2, as well as developing transgenic hairy roots with specific gene targets using the Crispr/cas9 approach and overexpression techniques. Furthermore, while the induced expression of all _LerHMGRs_ except _LerHMGR1_ and _LerHMGR2_ was very low, the expression of most genes in B5 to M9 followed a time-dependent pattern of first increasing, then decreasing, and then increasing, which was attributed to the stimulating effect during medium conversion. However, the expression of _LerHMGR2_ increased continuously throughout time, suggesting that _LerHMGR2_ may play a significant role in the biosynthesis of shikonin in response to various environmental and developmental factors. The molecular mechanism of the post-translational regulation of HMGR in _Arabidopsis_ has been extensively studied: the activity of HMGRIS and HMGR1L is negatively regulated by the phosphorylation of two B-type regulatory subunits of Ser/Thr protein phosphatase 2A (PP2A) phosphatase: B\({}^{\prime\prime}\)\(\alpha\) and B\({}^{\prime\prime}\)\(\beta\)[60; 61]. However, research on the transcriptional regulation of HMGR in higher plants is currently restricted. At the transcriptional level, the expression of _HMGR_ is regulated by proteins that bind to the sterol regulatory elements of the _HMGR_ promoter [62]. To further explore the transcription factors that may regulate the _HMGR_ transcript level, we first identified 2419 candidate transcription factors from the _L. erythrorhizon_ genome (Table S7), and then performed co-expression network analysis using all transcription factors, _LerHMGR1_ and _LerHMGR2_, based on transcriptomes data (Table S8). As shown in Figure S5, LE19274.1, LE10602.1, and LE00798.1, belonging to C2H2, and LE28791.1, belonging to AP2/ERF-ERF, had significant positive correlations with _LerHMGR1_ and _LerHMGR2_ (r > 0.65, \(p\) < 0.05), suggesting that they may be transcription factors that positively regulate the _HMGRs_. In conclusion, we thoroughly investigated the _HMGR_ gene family in 36 representative plants. Exploring the evolutionary history of the gene family and identifying the key school genes involved are a necessary basis for improving the shikonin biosynthesis pathway and increasing shikonin production through genetic engineering.