Despite the extensive application values of terpenoids, there are limitations in the stable and large-scale supply of terpenoids to meet all the demands of industry. Recent advances in metabolic engineering with the combination of synthetic biology and systems biology have enabled researchers to bring various cell factories [2, 4, 24], and several successful cases have been utilized in the biotech industry .
The increase of precursor supply is one of the effective strategies to achieve the flux enhancement for producing desired chemical compounds [20, 24, 26]. Overexpression of DXS, the first committing step in MEP pathway, has been frequently conducted to enhance the metabolic precursor pools for terpenoid production. This strategy, indeed, increased carbon flux and improved terpene production [27,28,29,30]. However, the following steps of the MEP pathway often hamper plant growth . In the case of E. coli, overexpression of DXS causes retarded growth and poor terpenoid production, which is attributed to the depletion of DXS substrates, glyceraldehyde 3-phosphate (G3P) and pyruvate [32,33,34]. Additionally, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECP), a MEP pathway intermediate, is known to be a limiting step due to its efflux from the E. coli cell. Activation of downstream MEP pathway enzyme reduced this efflux to effectively channel metabolite flux to the pathway end products, IPP and DMAPP . Moreover, MECP was reported as a retrograde-signaling metabolite, and it induced stress-related genes in Arabidopsis . In plants, removal of G3P from the Calvin cycle due to DXS-overexpression was also suggested to contribute to the unsatisfactory growth of the Arabidopsis transformant . It is possible, however, that overexpression of HDR would not draw G3P directly from Calvin cycle as opposed to overexpression of DXS, and thus positively contribute to the pool of isoprene units without critically disturbing carbon flux in the Calvin cycle. In addition, enhanced photosynthetic pigment contents due to the increased pool of isoprene units, in turn, would boost the photosynthetic rate to offset the partial siphoning of G3P from the Calvin cycle to the MEP pathway. For instance, the co-expression of tomato HDR and taxadiene synthase in Arabidopsis increases carotenoid and taxadiene levels . It was observed that increased HDR activity caused improved plastidial terpenoid biosynthesis in Arabidopsis . Therefore, we considered that overexpression of HDR that is downstream of MECP is thus a more rational approach to increase MEP pathway carbon flux by circumventing other negative cellular responses.
Generally in gymnosperm, HDR is known to present as isozymes, and our previous study also identified that G. biloba has a small family of three HDRs . In fact, each member of the HDR family has been suggested to have a distinct role in terpenoid biosynthesis. Class 1 HDRs, including GbHDR1are suggested to be involved in primary household metabolism whereas class 2 HDRs relate to secondary metabolism. Class 2 HDRs are expressed in both a developmental and spatial specific manner while no such specificities occur in class 1 HDRs (Additional file 1: Fig. S3) [15, 18, 38, 39]. Recently, we investigated the impact of class 2 GbHDR by overexpressing the GbHDR2 in tobacco plants and confirmed an increase of diterpenoid contents in GbHDR2ox tobacco plants . Therefore, we expected that the GbHDR1 overexpression study would demonstrate not only the enhancement of carbon flux through the MEP pathway but also the specific functions of GbHDR1, that are related to primary metabolism.
The unavailability of Ginkgo transformation system to date justified the use of a heterologous plant system, which would lead to an evaluation of the application potential of GbHDR1 in plant metabolic engineering. The poplar tree is an ideal plant model system with various advantages such as fast growth and high biomass. Wood composition of the plants is especially desirable in engineering plants for fuel uses. . In addition, the triploid poplar trees used in this study are free from biosafety issues because they prevent gene escape due to infertility. Furthermore, extensive poplar studies provide many accessible genetic tools and information to use the system straightforwardly for metabolic engineering research. Thus, we attempted to overexpress the GbHDR1 in poplar to acquire stable transgenic plants of GbHDR1ox. In the initial growth stage, the GbHDR1ox poplar plants exhibited rapid growth accompanied by increased height and leaf number compared with the WT plants (Fig. 2). GbHDR1ox poplar plants formed winter buds late compared to the WT poplars, which suggests high tolerance against low temperatures in GbHDR1ox poplars. Poplars temporally cease shoot growth to avoid permanent growth termination by forming winter buds to protect from low and freezing temperatures.
The enhanced growth of GbHDR1ox plants suggested the increased carbon flux towards several terpenoid metabolites such as isoprene, GA, chlorophylls and carotenoids. For example, an increase of IS transcript levels suggested increased C5 isoprene precursor pools in GbHDR1ox poplars (Fig. 3B). Additionally, up-regulation of IS transcription indirectly reflected enhanced isoprene emission from leaves. The emission of isoprene is known to help plants overcome abiotic stress such as drought and temperature by protecting photosynthesis . This fact suggests that the leaves of GbHDR1ox poplar can be an excellent platform to produce volatile terpenoids even under various stress conditions.
Enhanced GA (diterpene) biosynthesis in GbHDR1ox poplars could promote plant elongation, and increased levels of photosynthetic pigments such as chlorophylls (diterpene side chain) and carotenoids (tetraterpene) would stimulate photosynthesis. We thus analyzed the transcript levels of key genes in the biosynthetic pathway of these terpenoid metabolites as illustrated in Fig. 3A. The data in the present study clearly indicate that overexpression of GbHDR1 in poplar brought about up-regulation of downstream genes in a feed-forward activation manner (Fig. 3A).
Wille et al.  suggested that the MEP pathway and the production of plastidial pigments have a significant degree of coordination at the gene expression level in a feed-forward manner. Thus, overexpression of DXS and HDR leads to increased levels of chlorophylls and carotenoids [27, 37]. The phytol side chain, a diterpene of MEP pathway origin, is an essential component of light-harvesting pigments, chlorophylls a and b, that anchors the pigments to the thylakoid membrane . In the present study, overexpression of GbHDR1 in poplar evoked upregulation of CHS and CAO and also resulted in enhanced accumulation of chlorophylls a and b. Plant carotenoids derived from the MEP pathway play a crucial role in photosynthesis as accessory pigments . In GbHDR1ox transgenic poplar plants, we observed increased transcription levels of GbPSY, a key enzyme in carotenoid biosynthesis, with an concomitant increase of carotenoid contents.
It is evident in the present experiment that, besides simple law of mass action, up-regulation of downstream genes was operating in the GbHDR1ox plants to effectively drain the accumulated MEP pathway of end-products. During deetiolation of tomatoes, strong upregulation of HDR is accompanied by increased transcription of PSY . Therefore, enhanced carotenoid accumulation due to HDR overexpression in the present work resembles the PSY-activating mechanism in chloroplasts. The upregulation of chlorophyll and carotenoid biosynthetic genes in turn increased chlorophyll and carotenoid contents in transgenic plants. Such increase of photosynthetic pigment contents would enhance the photosynthesis rate. Indeed, overexpression of GbHDR1 led to elevated photosynthetic rates in the leaves of poplar (Fig. 4C).
Upregulation of KS and GA20ox transcription suggests elevated GA biosynthesis in the GbHDR1ox poplar plants (Fig. 3B). Because the biosynthesis of gibberellin is self-regulating, the effect of GbHDR1 overexpression on gibberellin-catabolizing GA2ox must be considered, as well . In this regard, it is very indicative that GbHDR1 overexpression in poplar resulted in the upregulation of KS and GA20ox with concomitant GA2ox downregulation (Fig. 3B). The results from both plants thus strongly imply increased active GA content in the GbHDR1ox plants. An increase in bioactive GA level is known to promote plant growth  and the inhibition of growth cessation under short-day photoperiod in poplar trees . Our data in GbHDR1ox poplars also displayed rapid growth and delayed terminal bud formation (Additional file 1: Fig. S1). Moreover, a previous GA20ox overexpression study in poplar proved that the increased bioactive GA level can improve the secondary growth in xylem fibers  and is consistent with our cross-section data of GbHDR1ox poplar that showed enhanced secondary xylem (Fig. 5 and Additional file 1: Fig. S2).
Increased photosynthesis and elevated GA levels are indicative of stem lengthening of stem in the GbHDRox1 plants. Recently, it was shown that the presence of the N-terminal region of the GbHDR1 enzyme is responsible for the shift of the optimal pH of the enzyme toward the basic side so the enzyme operates under alkaline condition of chloroplast stroma . Therefore, GbHDR1 best functions while the photosynthetic apparatus is operating. The close relationship of the enzyme with primary metabolism, embodied by expedited growth as shown in the present study, is suggestive.
Overexpression of GbHDR1 would be an applicable strategy to boost accumulation of the target terpenes especially in various plant cell platforms. Plant system as a cell factory platform have many advantages for engineering the complicated plant terpene metabolic pathways [24, 26, 40]. Additionally, they use light for photosynthesis and no externally added sugars are required to synthesize secondary metabolites, which enable eco-friendly and sustainable way of plant origin chemical production. In particular, terpenoid production as fuels, which require vast amount of biomass, would be economically viable when using a plant system like the poplar cell factory.