De novo induction of a DNA-histone H3K9 methylation loop on synthetic human repetitive DNA in cultured tobacco cells.

Genetic modifications in plants are crucial tools for fundamental and applied research. Transgene expression usually varies among independent lines or their progeny and is associated with the chromatin structure of the insertion site. Strategies based on understanding how to manipulate the epigenetic state of the inserted gene cassette would help to ensure transgene expression. Here, we report a strategy for chromatin manipulation by the artificial tethering of epigenetic effectors to a synthetic human centromeric repetitive DNA (alphoid DNA) platform in plant Bright-Yellow-2 (BY-2) culture cells. By tethering DNA-methyltransferase (Nicotiana tabacum DRM1), we effectively induced DNA methylation and histone methylation (H3K9me2) on the alphoid DNA platform. Tethering of the Arabidopsis SUVH9, which has been reported to lack histone methyltransferase activity, also induced a similar epigenetic state on the alphoid DNA in BY-2 cells, presumably by activating the RNA-dependent DNA methylation (RdDM) pathway. Our results emphasize that the interplay between DNA and histone methylation mechanisms is intrinsic to plant cells. We also found that once epigenetic modification states were induced by the tethering of either DRM1 or SUVH9, the modification was maintained even when the direct tethering of the effector was inhibited. Our system enables the analysis of more diverse epigenetic effectors and will help to elucidate the chromatin assembly mechanisms of plant cells.

Production of the bioactive plant-derived triterpenoid morolic acid in engineered Saccharomyces cerevisiae.

Morolic acid is a plant-derived triterpenoid that possesses pharmacological properties such as cytotoxicity, as well as anti-HIV, anti-HSV, anti-inflammatory, and antidiabetic effects. The significant therapeutic properties of morolic acid are desirable in the context of pharmacological and drug development research, but the low accessibility of morolic acid from natural resources limits its applications. In the present study, we developed a microbial system for the production of morolic acid. Using a combinatorial biosynthesis approach, a novel production pathway was constructed in Saccharomycescerevisiae by coexpressing BfOSC2 (germanicol synthase) from Bauhinia forficata and CYP716A49 (triterpene C-28 oxidase) from Beta vulgaris. Moreover, we reconstructed the cellular galactose regulatory network by introducing a chimeric transcriptional activator (fusion of Gal4dbd.ER.VP16) to overdrive the genes under the control of the galactose promoter. We also overexpressed truncated HMG1, encoding feedback-inhibition-resistant form of 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 and sterol-regulating transcription factor upc2-1, to increase the isoprenoid precursors in the mevalonate pathway. Using this yeast system, we achieved morolic acid production up to 20.7 ± 1.8 mg/L in batch culture. To our knowledge, this is the highest morolic acid titer reported from a heterologous host, indicating a promising approach for obtaining rare natural triterpenoids.

Identification of oxidosqualene cyclases from the medicinal legume tree Bauhinia forficata: a step toward discovering preponderant α-amyrin-producing activity.

Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography-mass spectrometry. Through transcriptome analyses, homology-based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α-amyrin synthase activity. B. forficata highly accumulated α-amyrin. We discovered an OSC with a preponderant α-amyrin-producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce ß-amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α-amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non-model legume plant.

Evidence that the Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-CoA reductase 1 is phosphorylated at Ser577 in planta.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in the mevalonate pathway. In higher plants, mevalonate pathway involves in the production of precursor for isoprenoids biosynthesis, including essential components for cell functions. Previously, we confirmed that the Arabidopsis thaliana HMGR1S (AtHMGR1S) is phosphorylated at S577 by the combination of sucrose non-fermenting related kinase-1 (SnRK1) and geminivirus rep-interacting kinase-1 (GRIK1) in vitro. However, even in quantitative phosphoproteomics studies that were directed to find SnRK1 target substrates, AtHMGR1S phosphorylation at S577 has never been detected in planta. In this study, we expressed AtHMGR1S as a C-terminal FLAG-fusion protein in A. thaliana hmg1 mutant to confirm its phosphorylation in planta. Our results provide the first direct evidence that AtHMGR1S is phosphorylated at S577 in planta. Moreover, phosphatase inhibitors treatment to the A. thaliana seedlings induced AtHMGR1S phosphorylation at sites other than S577, suggesting the presence of a novel HMGR regulatory mechanism in planta.

AKIN10, a representative Arabidopsis SNF1-related protein kinase 1 (SnRK1), phosphorylates and downregulates plant HMG-CoA reductase.

HMG-CoA reductase (HMGR) is a key enzyme in the mevalonate pathway for sterols and cytosolic isoprenoid production. Although HMGR kinases from spinach, barley, and cauliflower tissues have been strongly suggested as members of SNF1-related protein kinases 1 (SnRK1), the phosphorylation and inactivation of HMGR by plant SnRK1s has not been demonstrated. In this study, we elucidated that AKIN10, an Arabidopsis SnRK1, acts as an HMGR kinase. The recombinant AKIN10 phosphorylates and inactivates AtHMGR1S using recombinant GRIK1 as the AKIN10 activator. In contrast, AKIN10-GRIK1 fails to inactivate AtHMGR1S-S577A, suggesting that this is achieved through Ser577 phosphorylation. Moreover, phosphorylation is detected not only in AtHMGR1S but also in AtHMGR1S-S577A, suggesting the presence of a novel regulatory mechanism of plant HMGR.