Characterization and expression analysis of MATEs in Cannabis sativa L. reveals genes involving in cannabinoid synthesis

Abstract

Introduction

The annual dioecious herb Cannabis sativa L. (C. sativa) from Cannabis family accumulates terpenophenolic cannabinoids in glandular trichomes and flowers of female plants (Andre et al., 2016). Till now, over 113 cannabinoids have been isolated from C. sativa and many are derived from non-enzymatic decarboxylaion of their acidic forms by heat or UV irradiation (Güllck and Moller, 2020). The well-studied cannabinoids cannabigerol (CBG), Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) share the same biosynthetic precursors of olivetolic acid (OA) and geranyl diphosphate (GPP) (Güllck and Moller, 2020). Phenolic OA is condensed from one molecule of hexanoyl-CoA and three molecules of malonyl-CoA by coordinated work of olivetol synthase (OLS) (Taura et al., 2009) and olivetolic acid synthase (OAC) (Gagne et al., 2012) in cytosol. The acyl-activating enzyme 1 (AAE1) provides hexanoyl-CoA by converting C6-hexanoic acid which is derived from fatty acid pathway (Stout et al., 2012), while C10-isoprenoid GPP is synthesized by 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastid (Fellermeier et al., 2001). Aromatic prenyltransferase 4 (aPT4) from UbiA protein superfamily subsequently prenylates OA by GPP to produce cannabichromenic acid (CBGA) (Luo et al., 2019; Güllck et al., 2020), which is further oxidized to CBG. CBGA could be converted to Δ9-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA) by flavoproteins Δ9-tetrahydrocannabinolic acid synthase (THCAS) (Sirikantaramas et al., 2005) or cannabidiolic acid synthase (CBDAS) (Taura et al., 2007), respectively, and subsequently decarboxylated to THC or CBD by light or heat.

Low abundance in C. sativa and important pharmaceutical use evokes tremendous interests for heterologous biosynthesis of cannabinoids. A notable example is the de novo production of THCA and CBDA in yeast Saccharomyces cerevisiae system (Luo et al., 2019). Co-expression of OA synthetic pathway (CsAAE1, CsOAC and CsOLS) along with plastid-localization-signal-free CsaPT4, secretory-signal-free CsTHCAS or CsCBDAS in a GPP-overproducing yeast strain yielded THCA or CBDA, respectively. When those genes were transiently expressed in tobacco (Nicotiana benthamiana) cells, however, either OA, CBGA or THCA was severely glucosylated (Güllck et al., 2020). In addition to been easily glucosylated, CBGA and THCA are cytotoxic (Morimoto et al., 2007). Therefore, mechanisms might exist in C. sativa cells that protect cannabinoids from glucosylation and inhibit their cytotoxicity.

Multidrug and toxic compound extrusion proteins (MATEs) are known to translocate second metabolites, ions, and phytohormones intra and intercellularly, and are associated with exnobiotic efflux, aluminum detoxification and disease resistance (Upadhyay et al., 2019). Moreover, MATEs are widely distributed in prokaryotes and eukaryotes, including plants. YdhE was the first isolated MATE from E.coli as a multidrug efflux protein that confers bacteria drug resistance (Morita et al., 1998). Soon after, ALF, the first plant MATE from Arabidopsis thaliana (A. thaliana) (Diener et al., 2001) and many other plant MATEs were sequentially discovered (Wang et al., 2016; Santos et al., 2017; Wang et al., 2017; Huang et al., 2021). Most MATEs consist of about 400-700 amino acids with 12 transmembrane helices and a common MatE domain (pfam 01554), but no consensus sequence thus far has been found in all MATEs (Kusakizako et al., 2020). MATEs transport second metabolites into and out of the membranes through the employment of either Na⁺ or H⁺ electrochemical gradient (Hvorup et al., 2003). For instance, NtMATE1 and NtMATE2 that localize on the vacuolar membrane of tobacco root cells are believed to sequestrate alkaloid nicotine within the vacuole of the roots, then nicotine is transported to the above-ground organs of the plant in response to plant biotic stresses (Shoji et al., 2009). Similar function in alkaloid transportation is also found for tobacco MATEs Nt-JAT1 and Nt-JAT2, which localize on the vacuolar membrane of leaf cells (Morita et al., 2009). GhTT2, GhMATE12, GhMATE16 and GhMATE38 from Gossypium hirsutum L. localized on the tonoplast are proposed to translocate phenolic pro-anthocyanidins (Gao et al., 2016; Xu et al., 2019). VvAM1 and VvAM2 expressed in berry skins of Vitis vinifera and subcellularly localized on tonoplast are responsible of translocating flavonoid anthocyanins (Perez-Diaz et al., 2014). Thus, we hypothesis that MATEs from C. sativa might have the ability to bind and translocate terpenophenolic cannabinoids or corresponding biosynthetic intermediates, protecting them from glucosylation and inhibiting their cytotoxicity in C. sativa cells.

In this study, we identified 42 MATEs in C. sativa genome and characterized their physical-chemical properties, gene structure, motif composition and gene expression patterns. Furthermore, qRT-PCR verification and cis-acting elements analysis of CsMATEs that predominantly expressed in cannabinoids accumulation tissues were conducted. In addition, effects of UV-B on accumulation of cannabinoids and corresponding biosynthetic substrates as well as the expression of CsMATE23, CsMATE28 and CsMATE34 were studied. Together, our result indicates that CsMATE28 and CsMATE34 might be involved in biosynthesis of cannabinoids or its biosynthetic intermediates.

Materials and methods

Results

Discussion

MATEs have been identified in a variety of plant species, including 56 MATEs in Arabidopsis, 55 in rice (Wang et al., 2016). 67 in tomato (Santos et al., 2017), 64 in potato (Huang et al., 2021), 70 in Medicago truncatula (Wang et al., 2017), 72 in cotton (Xu et al., 2019) and 117 in soybean (Liu et al., 2016). In the present study, we identified 42 CsMATEs and investigated their physical-chemical properties, gene distribution, evolutionary relationships, conserved motifs, gene structures and gene expressions. CsMATEs contains only 42 members (Table 1) and is the fewest when compared with other plant species, which suggests CsMATEs may have undergone contraction during evolution. Those 42 CsMATEs were divided into four subfamilies and distributed on 10 chromosomes, with ~50% CsMATEs adjacent to at least one another CsMATEs (Figure 1), thus the expansion of CsMATEs might be largely due to tandem duplication (Wang et al., 2019).

Although cannabinoids synthetic pathways have already been illustrated, the translocation of the cannabinoids and biosynthetic intermediates are not known (Güllck and Moller, 2020). This inevitably hinders cannabinoids heterologous biosynthesis (Güllck et al., 2020). Cannabinoids are synthesized in glandular trichomes and flowers of female C. sative and their biosynthetic processes undergo cellular compartmentalization (Güllck and Moller, 2020). For instance, plastid localized CsaPT4 prenylates cytosolic synthesized OA in chloroplast to yield CBGA (Taura et al., 2009; Gagne et al., 2012; Luo et al., 2019; Güllck et al., 2020). Metabolite transporter proteins that could shuttle from cytosol to chloroplast should exist in C sativa cells. However, subcellular localization prediction indicates that vast majority of CsMATEs locate in vacuole or plasma membrane (Table 1). Although CsMATE39 contains putative chloroplast localization signal (Table 1), it expresses mainly in leaf (Figure 4A), the tissue that is not the most abundant cannabinoids accumulate. We assume that transporters from other families might be involved in translocating phenolic OA such as ATP binding cassette transporters which could translocate plant phenolic metabolites (Hwang et al., 2016). THCAS is secreted to apoplastic space of the glandular trichome to carry out oxidative cyclization of CBGA (Sirikantaramas et al., 2005). The plastid produced CBGA thus should be translocated across the plasm membrane. Thirty-five CsMATEs are predicted to localized on plasm membrane (Table 1). Of them, CsMATE03, CsMATE05, CsMATE23, CsMATE28, CsMATE31, CsMATE34 and CsMATE40 had a highest expression in flower but also expressed moderately in stem and leaf (the tissues that have glandular trichomes) according to transcriptome data (Figure 4A) and qRT-PCR (Figure 4B). Although we don’t have the trichome-specific transcriptional data of those seven genes in variety DK, they uniformly expressed in the trichomes of the other nine C. sativa varieties (Zager et al., 2019, Supplementary Figure S2 ). Moreover, the seven genes exhibited similar expression pattern in different tissues as that of cannabinoid synthetic genes (Figure 4A), which further indicates the involvement CsMATEs in biosynthesis of cannabinoids.

Within the seven genes, except for CsMATE23, CsMATE28, CsMATE34 and CsMATE40 all have an unexpressed alternative spliceform in tissues we studied (Figure 5A). Light responsive cis-acting elements were abundantly detected in promoters of the representative CsMATEs (CsMATE23, CsMATE28 and CsMATE34), reflects those genes could be regulated by lights. UV-B is known to affect accumulation of cannabinoids (Lydon et al., 1987). In accordance with this, the contents of cannabinoids and corresponding intermediates were changed under UV-B treatment (Figure 6A). As expected, the variation trend of CBGA was analogous to its direct downstream THCA, CBDA and CBG (Figure 6A), reflects the precursor nature of the CBGA (Güllck et al., 2020). Notably, although overall variation of OA is similar to CBGA, accumulation of OA is opposed to CBGA at two hours treatment where OA content was decreased but CBGA contents was increased compared to zero hours treatment (Figure 6A). Meanwhile, GPP did not show any change at two hours treatment (Figure 6A). Therefore, increase of CBGA after two hours UV-B treatment could be largely caused by over-usage of OA for synthesizing CBGA either due to enhanced CBGAS activity or more efficient translocation of OA form cytosol to plastid. Unfortunately, none of the CsMATEs we tested was up-regulated after two hours treatment (Figure 6B). We reasoned that plastid located ATP binding cassette transporters that translocate phenolic metabolites might responsible for OA transportation (Hwang et al., 2016). Moreover, Although the transcriptional variation of CsMATE23 is analogous to the content of OA (Figure 6B), OA is synthesized in cytosol and CsMATE23 is localized in plasma membrane (Table 1 ). Meanwhile, CsMATE23 is barely transcribed in C sativa root, however its Arabidopsis ortholog AtRHS2 (AtDTX31) located in plasma membrane of root is required for hairy root elongation (Won et al., 2009). Hence, we may also exclude participation of CsMATE23 in transporting OA precursors.

The most interesting finding we observed is the correlation between transcriptional alterations of CsMATEs with the accumulation of CBGA, THCA and CBDA (Figure 6). As the precursor of THCA and CBDA, CBGA content did not show much alteration at 12 hours treatment compared to the control zero hour (Figure 6A). However, the contents of THCA, THC, CBDA and CBD were all increased at least more than four times at 12 hours compared to zero hours and transcriptions of CsMATE28 and CsMATE34 were also significantly enhanced at 12 hours (Figure 6B). This could be explained by that increased CsMATE28 or CsMATE34 more inefficiently transport CBGA to the apoplastic space to enable its subsequent conversions (Sirikantaramas et al., 2005).

Conclusion

We identified 42 CsMATEs and analyzed their structural features, evolutionary relationships and expression patterns. We found number of CsMATEs was subjected to contraction and its expansion within the family was mainly due to tandem duplication. Though RNA-seq and qRT-PCR analysis, we found two root-specifically transcribed CsMATEs (CsMATE17 and CsMATE27) and three CsMATEs (CsMATE23, CsMATE28 and CsMATE34) whose transcription pattern were correlated with transcriptions of cannabinoids biosynthetic genes. In addition, although CBGA content was not much affected under UV-B treatment at 12 hours, accumulations of THCA, CBDA and CBG were increased. This could be due to the increased expression of CsMATE28 or CsMATE34. Although multiple evidences suggest that CsMATEs may play an important role in the synthesis and transport of cannabinoids, the transport of cannabinoids is a complex process. Therefore. The functions of candidate genes (CsMATE28 and CsMATE34) in cannabinoid transportations should be investigated in depth in the future.

Data availability statement

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

Author contributions

WC and HW designed the study. WC, HW, SW, XC and XM wrote the manuscript. SW, XC, XM, YW, AW, MA and QD conducted the bioinformatic analysis. XC and XM performed the qRT-PCR and UV-B treatment. All authors contributed to the article and approved the submitted version.

Funding

This work is supported by Scientific and technological innovation project of China Academy of Chinese Medical Sciences (CI2021A04113).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

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References