Cross-talk between the methylerythritol phosphate and

14 Plants use two distinct isoprenoid biosynthesis pathways: the methylerythritol phosphate 15 (MEP) pathway and the mevalonic acid (MVA) pathway. 1-deoxy-D-xylulose5-phosphate 16 synthase (DXS) and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) are the 17 rate-limiting enzymes in the MEP pathway, and 3-hydroxy-3-methylglutaryl-CoA reductase 18 (HMGR) is a key regulatory enzyme in the MVA pathway. Previously, overexpression of 19 Populus trichocarpa PtDXR in Nanlin 895 ( Populus × euramericana cv. ‘Nanlin 895’) poplar 20 was found to upregulate resistance to salt

The MVA pathway occurs in the cytoplasm, ER, as well as peroxisomes and produces sesquiterpenoids and sterols.3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), which is a rate-limiting enzyme in the MVA pathway can catalyze 3-hydroxy-3-methylglutary-CoA (HMG-CoA) to form MVA (Cowan et al., 1997;Roberts et al., 2007).In addition, the MEP pathway occurs in the chloroplast and is responsible for producing carotenoids, ABA, GAs, and diterpenoids.
Terpenoids in plant cells are involved in diverse cellular processes.Sesquiterpene derivatives such as phytoalexin and volatile oils play important roles in plant growth, development, and disease resistance (Hain et al., 1993;Ren et al., 2008).α-Tocopherol (vitamin E) is an antioxidant that improves human health (Weber et al., 1997).Sterols are an important component of biological membranes, which are linked to metabolism (Huang et al., 2008).
Photosynthetic pigments participate in plant photosynthesis and enable the conversion of basic carbon sources for plant growth (Esteban et al., 2015).Plant hormones (such as ABA and GAs) are connected to plant growth and the responses to biotic and abiotic stresses (Kazan et al., 2015).Paclitaxel is one of the most effective chemotherapy agents for cancer treatment, and artemisinin is an anti-malarial drug (Kong et al., 2015;Kim et al., 2016).
Previous metabolic engineering studies have proposed strategies for improving the yields of specific metabolites in plants.For example, the industrial exploitation of hairy roots has been used to enhance secondary metabolite production (Zhang et al., 2004;Rahman et al., 2009).3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is a second enzyme that has been genetically engineered to improve terpenoid content.For example, overexpression of Brassica juncea HMGS in tomato plants leads to upregulated carotenoid and phytosterol levels (Liao et al., 2018).HMGR can be metabolically engineered to regulate the content of terpenoids (Aharoni et al., 2005;Dueber et al., 2009).In Arabidopsis thaliana, HMGR1 has been shown to play an important role in the biosynthesis of sterols and triterpenoids through investigation of A. thaliana mutant lines (Suzuki et al., 2005).Moreover, overexpression of PgHMGR1 from ginseng in A. thaliana can lead to increased production of sterols and triterpenes (Kim et al., 2014).Transgenic tobacco overexpressing the HMGR of Hevea brasiliensis has elevated levels of phytosterol (Schaller et al., 1995).Dai et al. (2011) expressed SmHMGR2 in Salvia miltiorrhiza, resulting in the improvement of squalene and tanshinone contents.Interestingly, transgenic T 0 tomato fruits display elevated levels of phytosterol with overexpression of HMGR1 from A. thaliana, but only certain phytosterols increase in mature T 2 fruits (Enfissi et al., 2005).DXR can be genetically engineered to regulate the content of terpenoids and expressed DXR in Arabidopsis and observed enhanced flux through the MEP pathway (Carretero-Paulet et al., 2006).Overexpression of A. thaliana DXR in Salvia sclarea hairy roots increases levels of anthiolimine, a diterpene (Vaccaro et al., 2014).Overexpression of DXR in peppermint affects the regulation of monoterpenes in leaf tissues (Mahmoud et al., 2001).Farnesyl diphosphate is the synthetic precursor of numerous primary metabolites including steroids, polyterpenoid alcohols, and ubiquinone, and represents the branching point of many terpenoid metabolic pathways (Wong et al., 2018).In summary, overexpression of genes in the MEP and MVA pathways through metabolic engineering can change the expression levels or activities of related enzymes and metabolic products, which opens up a new direction for plant breeding and acquisition of metabolic products.
To date, most studies have found that the MVA and MEP pathways are located in different parts of plants.The MVA pathway occurs in the cytoplasm and endoplasmic reticulum and involves the biosynthesis of secondary metabolites such as sterols, sesquiterpenes, and triterpenes.Meanwhile, the MEP pathway takes place in plastids and mainly involves the biosynthesis of diterpenes, monoterpenes, carotenoids, and isoprene.Previous studies have shown that the MVA and MEP pathways do not operate in isolation and that some metabolic intermediates that link them can be exchanged through plastid membranes (Laule et al., 2003;Liao et al., 2006).In this study, to comprehensively investigate such cross-talk in poplar, we determined the transcript levels of MEP-and MVA-related genes in PtHMGR-OEs and PtDXR-OEs.The results showed that not only were the transcript levels of MVA-related genes (such as acetoacetyl CoA thiolase [AACT], mevalonate kinase [MVK], and mevalonate

5-diphosphate decarboxylase [MVD]
) upregulated in PtHMGR-OEs, but also the expression levels of MEP-related genes (such as DXS, DXR, HDS, and HDR) were improved.In PtDXR-OEs, the transcript levels of MVA-related genes were downregulated, while the expression levels of MEP-related genes were increased.Based on these results, we propose that the MEP pathway is dominant and the MVA pathway is subordinate.Overexpression of PtDXR in poplar increases the expression levels of MEP-related genes, which may result in the accumulation of terpenoids, and then feedback with the MVA pathway may lead to the inhibited expression of MVA-related genes.In addition, the transcript levels of MEP-related genes and products, such as MEP-derived isoprenoids, were affected in PtHMGR-OEs.These results demonstrate that cross-talk occurs between the MEP and MVA pathways.
In addition, the results of Southern blotting indicated a copy number of at least three copies for PtHMGR in the poplar genome (Figure 1A).As shown in Figure 1B, probe 2 could specifically recognize PtHMGR (Potri.004G208500.1),indicating that the probe 2 used for qRT-PCR of PtHMGR could be applied to the follow-up study of PtHMGR expression levels.
Based on the whole genome of 10 species achieved from Phytozome, an evolution tree was constructed and showed the evolutionary relationship of those species (Figure 2A).Also, the phylogenetic tree illustrated that the function and evolution of the HMGR family are relatively strongly conserved among all plants (Figure 2B).Also, the HMGR originated from a single ancestor and evolved into two different groups, one each in monocotyledons and dicotyledons (Ferrero et al., 2015;Li et al., 2014).This result shows that the evolutionary tree constructed from HMGR is consistent with that constructed from the whole genomes of various species (Figure 2).
Analyses of gel electrophoresis illustrated that the PtHMGR gene was inserted into the genome of "Nanlin 895" plants obtained through screening with kanamycin (Kan) using the primer for the cauliflower mosaic virus (CaMV) 35S promoter (Supplemental Table 1) as the forward primer and that for PtHMGR-R (Supplemental Table 1) as the reverse primer (Supplemental Figure 1).Besides, the transcript levels of PtHMGR were higher in transgenic lines than in NT plants, showing that PtHMGR could be stably expressed in Nanlin 895 plants (Supplemental Figure 2).Overall, PtHMGR was inserted into poplar chromosomes and expressed in poplar cells.

PtDXR overexpression affects expression levels of MVA-and MEP-related genes in transgenic poplars
When determining the transcript levels of MVA-and MEP-related genes in PtDXR-OE1-1 and PtDXR-OE3-1 via qRT-PCR, we found that the expression levels of DXS, HDS, HDR, MCT, and CMK genes involved in the MEP pathway were significantly greater in transgenic poplars.Notably, MVA-related genes (AACT, HMGS, HMGR, MVK, MVD) were downregulated in PtDXR-OE seedlings (Figure 4), the opposite of the MVA-related gene expression results obtained from PtHMGR-OEs.In addition, the expression levels of FPS, IDI, GPS, and GPPS were significantly elevated in both PtDXR-OE1-1 and PtDXR-OE3-1 compared to the NT poplars (Figure 4).ZEP3 in PtHMGR-OE poplars were elevated compared to that in NT poplars (Figure 3).In addition, ABA levels were significantly elevated in lipid extracts from HMGR-OE poplar leaves (Figure 5).The contents of β-carotene and lycopene, which are intermediate products in the synthesis of ABA, were identified via HPLC, and the results showed significant enhancement of each in HMGR-OE leaves (Figure 5) Lutein content was also significantly elevated in HMGR-OE leaves (Figure 5).In addition, the total level of GA3, a downstream product of MEP, increased to 0.2-0.35ng/g in PtHMGR-OE leaves, compared to 0.08-0.1 ng/g in NT leaves (Figure 6A).Among MVA-related factors, the results of HPLC-MS/MS showed that the TZR and IPA contents were higher in PtHMGR-OEs than in the NT (Figure 6B, and C).

Overexpression of
The DCS content of PtHMGR-OEs increased to 0.31-0.41ng/g relative to 0.74-1.12ng/g in the NT (Figure 6D), representing an average 3-fold increase in PtHMGR-OEs.However, the CS content showed the opposite result in the comparison between PtHMGR-OEs and NT (Figure 6E).Together, these results illustrate that the HMGR gene not only causes changes in MVA-related genes and their products but also affects MEP-related genes and products.
2.5.Enhanced TZR, IPA, DCS, carotenoid, ABA, and GA3 levels in PtDXR-OE poplars The levels of β-carotene, lycopene, and ABA were much higher in PtDXR-OE poplars than in NT plants, and lutein, which is considered a MEP-derived hormone, showed a similar result (Figure 7).When the molecular mechanism underlying the improvement of ABA in PtDXR-OEs was investigated, the expression levels of NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and ZEP3 were significantly higher in PtDXR-OEs than in NT plants (Figure 7).
In addition, a significant increase in GA3 in PtDXR-OEs was observed through HPLC-MS/MS analyses (Figure 8A).In addition to upregulating MEP-derived products, overexpression of PtDXR also affected the contents of MVA-derived products.The TZR content of PtDXR-OEs increased to 0.24-0.37ng/g compared to 0.0274-0.033ng/g in NT plants (Figure 8B), representing an average 10-fold increase in PtDXR-OEs.The content of IPA in PtDXR-OEs increased to 0.84-1.12ng/g, compared to 0.32-0.41ng/g in NT poplars (Figure 8C), representing an average 3-fold increase in PtDXR-OEs.The DCS content of PtDXR-OEs was also significantly higher.Its level increased to 3.06-3.62ng/g, with only 1.47-1.52ng/g in NT plants (Figure 8D), representing an average 3-fold increase in PtDXR-OEs (Figure 8D).By contrast, the content of CS in PtDXR-OEs significantly decreased compared to that in NT poplars (Figure 8C), indicating significant downregulation in PtDXR-OEs (Figure 8E).We were unable to determine the contents of zeatin, IP, GA1, GA4, and GA7 through HPLC-MS/MS (Supplemental Figure 15).

Characterization and evolutionary history of HMGR
Previous studies have shown that NtHMGR2 is a stress-responsive gene, whereas NtHMGR1 is a housekeeping gene (Hemmerlin et al., 2004;Merret et al., 2007).LcHMGR1 is most highly expressed during the early stages of fruit development and is involved in the regulation of fruit size.The level of LcHMGR1 expression is higher and lasts longer in larger fruits, while LcHMGR2 is expressed most highly during the late stages of fruit development and is related to the biosynthesis of isoprenoid substances required for cell elongation during that time (Rui et al., 2012).The expression of CaHMGR1 is temporary and tissue-specific, whereas that of CaHMGR2 is constitutive.CaHMGR1 is only expressed in fruit tissues (pulp, endosperm, and endocarp), flower buds, and leaves during the initial stages of development, while CaHMGR2 is expressed in all tissues (flower buds, leaves, branches, and roots) and fruit tissues at various stages of development (Tiski et al., 2012).In this study, we found that there is a copy number of at least three for PtHMGR genes in the poplar genome and that the PtHMGR gene (Potri.004G208500.1)has high homology with other known HMGR1 genes based on multiple alignment analyses.In addition, the whole genome and HMGR protein sequences of various species were used to construct evolutionary trees, and the resulting evolutionary relationships among species were similar.Because HMGR in plants is a relatively conserved gene in the MVA pathway, it should be considered an important factor in studies of biological genetic differentiation and molecular evolution, and can be used as a reference for determining relationships among species.In addition, overexpression of BjHMGS1 in tomato causes the expression levels of GPS and GPPS to significantly increase (Liao et al., 2018).In the present study, we also investigated the expression levels of FPS, GPS, and GPPS, and overexpression of PtHMGR increased their expression levels, which may have enhanced the interaction between IPP and dimethylallyl diphosphate (DMAPP), thereby increasing the biosynthesis of plastidial C15 and C20 isoprenoid precursors.

HMGR regulates isoprenoid biosynthesis genes in
Xu et al 46 showed that overexpression of the HMGR gene in Ganoderma lucidum causes no significant changes in farnesyl diphosphate synthase (FPS), squalene synthase (SQS), or lanosterol synthase (LS) mRNA expression, despite increasing the contents of squalene and lanosterol.Overexpression of BjHMGS1 in tomato significantly increases transcript levels of FPS, SQS, squalene epoxidase (SQE), and cycloartenol synthase (CAS) (Liao et al., 2018).
Notably, the transcript levels of AACT, MVK, and MVD are significantly upregulated in PtHMGR-OE poplars although the expression level of HMGS is reduced.The increased expression has been observed for most MVA-related genes that contribute to the biosynthesis of sesquiterpenes and other C15 and universal C20 isoprenoid precursors.TwHMGS, TwHMGR, TwFPS, and TwGPPS, but decreases that of TwDXS (Zhang et al., 2018).

Overexpression of
Overexpression of NtDXR1 in tobacco increases the transcript levels of eight MEP-related genes, indicating that overexpression of NtDXR1 could lead to an upregulated expression of downstream genes in the MEP pathway (Zhang et al., 2015).Changes in the DXR transcript level of transgenic A. thaliana do not affect DXS gene expression or enzyme accumulation, although overexpression of DXR boosts MEP-derived isoprenoids such as carotenoids, chlorophylls, and taxadiene in A. thaliana (Carretero-Paulet et al., 2015).Overexpression of the potato DXS gene in A. thaliana can cause upregulation of the downstream GGPPS gene and PSY (phytoene synthase) gene (Henriquez et al., 2016).In addition, the transformation of the DXS gene from A. thaliana into Daucus carota significantly enhances the expression level of the PSY gene (Simpson et al., 2016).
Our results show both similarities and differences compared to previous studies.For example, we found that the transcript levels of MEP-related genes were higher in PtDXR-OE poplars than in NT poplars.However, overexpression of PtDXR in poplar led to significantly downregulated expression levels of MVA-related genes.Together, these findings illustrate that carbon sources may be preferentially synthesized into monoterpenes, diterpenes, and tobacco, and it increased the sterol content of transgenic tobacco as well as the accumulation of intermediate metabolites (Schaller et al., 1995).In another study, A. thaliana HMGR overexpression in Lavandula latifolia increased the levels of sterols in the MVA pathway and of MEP-derived monoterpenes and sesquiterpenes (Munoz-Bertomeu et al., 2007).In still another, overexpression of the SmHMGR gene in hairy roots increased MEP-derived diterpene tanshinone (Kai et al., 2011).In our study, ABA synthesis-related genes (NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and ZEP3) and the contents of ABA, GA3, and carotenoids were upregulated in PtHMGR-OE poplar seedlings, suggesting that overexpression of HMGR may indirectly affect the biosynthesis of MEP-related isoprenoids including ABA, GAs, and carotenoids.The accumulation of MVA-derived isoprenoids including TZR, IPA, and DCS was significantly elevated in PtHMGR-OEs, indicating that PtHMGR overexpression has a direct influence on the biosynthesis of MVA-related isoprenoids.Therefore, the HMGR gene not only directly affects the contents of MVA-derived isoprenoids but also indirectly affects the content of MEP-derived isoprenoids by changing the expression levels of MEP-related genes.
3.5.Upregulation of MEP-derived products (ABA, GA3, and carotenoid) and MVA-derived products (TZR, IPA, DCS) in PtDXR-OE seedlings The production of MEP catalyzed DXR, which is the most important rate-limiting enzyme in the MEP pathway and an important regulatory step in the cytoplasmic metabolism of isoprenoid compounds (Takahashi et al., 1998).In Mahmoud et al. (2001), overexpression of DXR in Mentha piperita promoted the synthesis of monoterpenes such as mint oil in the leaves and increased the production of essential mint oil by 50%.In Hasunuma et al. (2008), overexpression of Synechocystis sp.strain PCC6803 DXR in tobacco resulted in increased levels of β-carotene, chlorophyll, antheraxanthin, and lutein.In another study, A. thaliana DXR mutant plants exhibited a lack of GAs, ABA, and photosynthetic pigments, and the phenotype of the mutant A. thaliana plants showed pale sepals and yellow inflorescences (Xing et al., 2010).In our study, ABA synthesis-related genes were dramatically increased, as was the accumulation of ABA, GA3, and carotenoids, in PtDXR-OE poplar seedlings, indicating an effect from DXR overexpression.Combined with the result described above of increased DXS, HDS, HDR, MCT, CMK, FPS, GPS, and GPPS expression levels, we can conclude that overexpression of DXR can directly affect the expression levels of MEP-related genes and that changes in MEP-related gene expression levels contribute to the field of ABA, GA3, and carotenoids.Although the expressions of AACT, HMGS, HMGR, MVK, and MVD, which are related to the MVA pathway, were downregulated, the contents of the MVA-derived components TZR, IPA, and DCS were upregulated in PtDXR-OE seedlings, aside from their reduced CS content.

Cross-talk in the MVA and MEP pathways
Although the starting materials differ between the MVA and MEP pathways, IPP and DMAPP are common precursors.Blocking only the MVA or the MEP pathway does not completely block the biosynthesis of terpenes in the cytoplasm or plastids, indicating that the common products of the MVA and MEP pathways can be transported freely to different subcellular regions (Aharoni et al. 2003;Aharoni et al. 2004;Gutensohn et al. 2013).For example, results of green fluorescent protein fusion and confocal microscopy have shown that HDR is localized in the chloroplast, and the transfer of IPP from the chloroplast to cytoplasm can be observed through 13 C labeling, indicating that plentiful IPP is available for use in the MVA pathway to produce terpenoids (Ma et al. 2017).In addition, segregation between the MVA and MEP pathways is limited, and some metabolites can be exchanged on the plasmid membrane (Laule et al., 2003).Kim et al. (2016) used clustered regularly interspaced short palindromic repeats (CRISPR) technology to reconstruct the lycopene synthesis pathway and control the flow of carbon in the MEP and MVA pathways.The results showed that MVA-related genes were reduced by an average of 81.6% and the lycopene yield was significantly boosted.According to analyses of gene expression levels and metabolic yield in PtHMGR-OEs and PtDXR-OEs, we found cross-talk not only between IPP and DMAPP in the MVA and MEP pathways but also between pairs of genes and between products of the MVA and MEP pathways in poplar cells.On one hand, overexpression of PtDXR affected the transcript levels of MEP-related genes and the contents of MEP-derived isoprenoids including ABA, GAs, and carotenoids.However, the decreased accumulation of MVA-related gene products reduces the yields of MVA-derived isoprenoids including CS, while increasing TZR, IPA, and DCS contents.We also found that differential expression of MVA-related genes in PtDXR-OE or post-transcriptional/post-translational regulation of the products of MVA-derived isoprenoids may have occurred.Furthermore, although the expression levels of MVA-related genes in PtDXR-OEs were lower than those in NT poplars, the transcript levels of IDI were upregulated in PtDXR-OEs, indicating that IPP and DMAPP produced by the MEP pathway could enter the cytoplasm to compensate for the lack of IPP and DMAPP there.On the other hand, PtHMGR-OE plants exhibited higher transcript levels of AACT, MVK, and MVD as well as higher expression levels of DXS, DXR, HDS, and HDR compared to NT, which indicates that PtHMGR affects both MEP-and MVA-related gene expression levels.We successfully demonstrated that manipulation of HMGR in the MVA pathway of poplars results in dramatically enhanced yields of ABA, GAs, and carotenoids.This result illustrates that cytosolic HMGR overexpression could lead to a boost in plastidial GPP-and GGPP-derived products, including ABA, GAs, and carotenoids.Therefore, this study provides evidence that cross-talk between the MVA and MEP pathways increased the expression levels of GPS and GPPS in PtHMGR-OEs, and elevated the contents of ABA, GA3, and carotenoids.Moreover, changes in MEP-and MVA-related gene expression levels affect MVA-and MEP-derived isoprenoids.Together, these results illustrate the phenomenon of cross-talk between the MVA and MEP pathways.The mechanism underlying this exchange between the MVA and MEP pathways requires further research.
In conclusion, Isoprenoid compounds are diverse, with complex structures and different properties, and play an important role in the survival and growth of plants.The basic framework of the isoprenoid metabolic pathway has been explored, and many advances have been made in clarifying the molecular regulation of metabolic pathways and metabolic engineering.However, previous studies have been limited to Arabidopsis, tomato, and rice, and research on isoprenoid metabolism in perennial woody plants has just begun.In this study, the overexpression of PtHMGR in Nanlin 895 (Populus× euramericana cv.'Nanlin 895') caused the accumulation of MVA-derived isoprenoids as well as MEP-derived substances including ABA, GAs, carotenoids, and GRs, which are essential plant hormones regulating growth and stress responses.In PtHMGR-OE poplars, most MEP-and MVA-related genes associated with the biosynthesis of isoprenoid precursors were upregulated.In PtDXR-OE poplars, elevated contents of ABA, GAs, carotenoids, and GRs were attributed to increased expression of MEP-related genes as well as plastidial GPP and GGPP.Moreover, in both PtDXR-OE poplars, changes in MVA-related genes caused changes in MVA-derived isoprenoids, including the yields of CKs and BRs.Together, these results show that the manipulation of PtDXR and PtHMGR is a novel strategy to influence poplar isoprenoids and provide a theoretical basis for the cultivation of high-quality poplar.

PtHMGR gene isolation and vector construction
Total RNA was extracted from P. trichocarpa leaves and reverse transcriptase (TaKaRa, Japan) was used to amplify the cDNA.Forward and reverse primers (Supplemental Table : PtHMGR-F and PtHMGR-R) were designed and polymerase chain reaction (PCR) was performed to synthesize the open region frame (ORF) of the PtHMGR gene.Subsequently, the product of the PtHMGR gene was ligated into the PEASY-T3 vector (TransGen Biotech, China) based on blue-white spot screening and the PtHMGR gene was inserted into the vector pGWB 9 (Song et al., 2016) using Gateway technology (Invitrogen, USA).
To identify the copy number of PtHMGR in P. trichocarpa, total genomic DNA was extracted from leaves according to the cetyltrimethylammonium bromide (CTAB) method and Southern blotting was performed.The poplar DNA was digested with EcoRI at 37°C for 4 h and the digested poplar DNA was separated on a 0.8% agarose gel at 15 V. Then the separated poplar DNA was transferred to a Hybond N + nylon membrane and blotting was performed according to the manufacturer's instructions (Roche, Basel, Switzerland).The details of probe 1 and probe 2 used for Southern blotting are provided in Supplemental Table 1.

Creation of phylogenetic tree
To cluster families based on protein-coding genes, the protein data for 10 plant species were downloaded Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html):P. trichocarpa and Zea mays PH207 (v1.1).Only the gene model for each gene locus that encodes the longest protein sequence was retained to remove redundant sequences.Then we employed DIAMOND software to identify potentially orthologous gene families among the filtered protein sequences of these species using a maximum e-value of 1e-5 (Buchfink et al., 2015).We used the OrthoFinder (v2.3.3)pipeline to identify single-copy gene families based on the aligned results with the default parameters (Emms et al., 2015).
We applied MAFFT (v7.158b) software (Katoh and Standley, 2013) for multiple sequence alignment of protein-coding sequences for each single-copy gene using the accurate option (LINS-i).Meanwhile, trimAl (v1.2) software (Capella-Gutiérrez et al., 2009) was employed to remove poorly aligned positions and divergent regions with the default parameters, and the alignments of all of the single-copy genes were concatenated to form a supermatrix.We used the program RAxML (v8.2.12) (Stamatakis, 2014) with the JTT+G+F model and 1000 bootstrap replicates to construct the supermatrix phylogenetic tree.Finally, the phylogenetic tree was visualized using FigTree (v1.4.3) (Drummond et al., 2009).The amino acid sequences of HMGR from P. trichocarpa, M. esculenta, S. purpurea, T. cacao, G. hirsutum, D. persica, M. domestica, A. thaliana, and Z. mays were obtained from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/) and Phytozome and MEGA5.0 software was used to construct a phylogenetic tree using 1000 bootstrap replicates.

Generation and characterization of transgenic poplars
Agrobacterium EHA105 containing a recombinant plasmid was used for infection of Nanlin 895 (Populus × euramericana cv.'Nanlin 895') leaves and petioles (Movahedi et al., 2014;Movahedi et al., 2020).Poplar buds were screened on differentiation MS medium amended with 30 μg/mL Kan and the resistant buds were planted in bud elongation MS medium containing 20 μg/mL Kan and transplanted into 1/2 MS medium containing 10 μg/mL Kan to generate resistant poplar trees.Subsequently, the DNA and RNA of putative transgenic poplars were isolated and analyzed via PCR and quantitative reverse transcription PCR (qRT-PCR).

Analyses via qRT-PCR
PtDXR-OE poplars have been obtained previously (Xu et al., 2019), and in this study, we extracted RNA from 12-month-old PtDXR-OE and PtHMGR-OE poplars.qRT-PCR was performed to identify the expression levels of MVA-and MEP-related genes in NT, PtDXR-OE, and PtHMGR-OE poplars.qRT-PCR was performed with a StepOne Plus Real-time PCR System (Applied Biosystems, USA) and SYBR Green Master Mix (Roche, Germany).Poplar Actin (PtActin) (XM-006370951.1) has previously been tested as an internal control for estimating the amount of RNA in samples (Zhang et al., 2013).The following conditions were used for qRT-PCR reactions: pre-denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and a chain extension at 60°C for 1 min.Three independent experiments were conducted using gene-specific primers (Supplemental Table 1).
PtHMGR leads to accumulation of MVA-derived TZR, IPA, and DCS as well as MEP-derived carotenoids, ABA, and GAs Analyses of carotenoids, ABA, GAs, zeatin, TZR, IPA, CS, and DCS, which are involved in plant growth and development as well as the responses to biotic and abiotic stresses, would help to elucidate plant growth and the stress response process and could guide poplar breeding.The HPLC-MS/MS chromatograms of ABA, GA, zeatin, TZR, IP, IPA, CS, and DCS standards are provided in Supplemental Figures3-13.The expression levels of ABA synthesis-related genes including 9-cis-epoxycarotenoid dioxygenase (NCED) and zeaxanthin epoxidase (ZEP) in HMGR-OE and NT leaves were analyzed via qRT-PCR, and ABA contents of PtHMGR-OE and NT leaves were analyzed via HPLC-MS/MS.The transcript levels of NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and PtHMGR-OE plantsLiao et al. (2018) showed that overexpression of BjHMGS1 affects the expression levels of MEP-and MVA-related genes and slightly increases the transcript levels of DXS and DXR in transgenic plants.However, the expression levels of DXS, DXR, HDS, and HDR are significantly improved in PtHMGR-OE poplars, while those of MCT and CMK are downregulated.
PtDXR affects MEP-and MVA-related genes in PtDXR-OE plants Overexpression of TwDXR in Tripterygium wilfordii increases the expression levels of tetraterpenoids in poplar cells.Secondary metabolic pathways in plants are tightly regulated by the transcript levels of MEP-and MVA-related genes.The dominant isoprenoid compounds synthesized in different plants differ greatly.The regulation of the expression of multiple enzymes largely controls whether specific secondary metabolites are synthesized and to what extent.The diversity of biosynthetic pathways, the complexity of metabolic networks, and the unknown regulation of gene expression lead to very different regulation patterns of MEP-and MVA-related gene expression among species.One possible conclusion is that MEP-and MVA-related genes often do not work alone, but instead are co-expressed with upstream and downstream genes in the MEP and MVA pathways to carry out a specific function.3.4.Overexpression of HMGR promotes the formation of ABA, GAs, and carotenoids in plastids and accumulation of TZR, IPA, DCS in the cytoplasm Plant HMGR, as the rate-limiting enzyme in the MVA pathway, plays a very important role in controlling the flow of carbon in that metabolic pathway.The upregulation of the HMGR gene significantly increases isoprenoid levels in plants.Studies on homologous and heterologous overexpression of HMGR have reported significantly elevated levels of isoprenoid in transgenic plants.In one study, Hevea brasiliensis HMGR1 was transferred into

Figure 1 .
Figure 1.Southern blotting analyses of homologous PtHMGR copy number and identification of probes specific to PtHMGR (Potri.004G208500.1).(A) Southern blotting was performed to determine the copy number of homologous PtHMGR genes using probe 1.(B) Southern blotting was performed using probe 2 to specifically recognize PtHMGR (Potri.004G208500.1).

Figure 3 .
Figure 3. qRT-PCR analyses of the transcript levels of MEP-and MVA-related genes in PtHMGR-OEs.Transcript levels of MEP-related genes including DXS, DXR, MCT, CMK, HDS, and HDR; of MVA-related genes including AACT, HMGS, MVK, and MVD; and downstream genes including IDI, GPS, GPPS, and GGPPS.PtActin was used as an internal

Figure 7 .
Figure 7. qRT-PCR analyses of the expression levels of ABA synthesis-related genes and HPLC-MS/MS analyses of the contents of ABA, β-carotene, lycopene, and lutein in PtDXR-OEs and NT poplars.The expression levels of ABA synthesis related genes including NCED1, NCED3, NCED5, NCED6, ZEP1, ZEP2, and ZEP3 are shown.Vertical bars represent mean ± SD (n = 3).*significant difference at P < 0.05.**significant difference at P < 0.01.***significant difference at P < 0.001.Three independent experiments were performed in this study.