A Cotyledon-based Virus-Induced Gene Silencing (Cotyledon-VIGS) approach to study specialized metabolism in medicinal plants.

BACKGROUND: Virus-induced gene silencing (VIGS) is widely used in plant functional genomics. However, the efficiency of VIGS in young plantlets varies across plant species. Additionally, VIGS is not optimized for many plant species, especially medicinal plants that produce valuable specialized metabolites. RESULTS: We evaluated the efficacy of five-day-old, etiolated seedlings of Catharanthus roseus (periwinkle) for VIGS. The seedlings were vacuum-infiltrated with Agrobacterium tumefaciens GV3101 cells carrying the tobacco rattle virus (TRV) vectors. The protoporphyrin IX magnesium chelatase subunit H (ChlH) gene, a key gene in chlorophyll biosynthesis, was used as the target for VIGS, and we observed yellow cotyledons 6 days after infiltration. As expected, the expression of CrChlH and the chlorophyll contents of the cotyledons were significantly decreased after VIGS. To validate the cotyledon based-VIGS method, we silenced the genes encoding several transcriptional regulators of the terpenoid indole alkaloid (TIA) biosynthesis in C. roseus, including two activators (CrGATA1 and CrMYC2) and two repressors (CrGBF1 and CrGBF2). Silencing CrGATA1 led to downregulation of the vindoline pathway genes (T3O, T3R, and DAT) and decreased vindoline contents in cotyledons. Silencing CrMYC2, followed by elicitation with methyl jasmonate (MeJA), resulted in the downregulation of ORCA2 and ORCA3. We also co-infiltrated C. roseus seedlings with TRV vectors that silence both CrGBF1 and CrGBF2 and overexpress CrMYC2, aiming to simultaneous silencing two repressors while overexpressing an activator. The simultaneous manipulation of repressors and activator resulted in significant upregulation of the TIA pathway genes. To demonstrate the broad application of the cotyledon-based VIGS method, we optimized the method for two other valuable medicinal plants, Glycyrrhiza inflata (licorice) and Artemisia annua (sweet wormwood). When TRV vectors carrying the fragments of the ChlH genes were infiltrated into the seedlings of these plants, we observed yellow cotyledons with decreased chlorophyll contents. CONCLUSIONS: The widely applicable cotyledon-based VIGS method is faster, more efficient, and easily accessible to additional treatments than the traditional VIGS method. It can be combined with transient gene overexpression to achieve simultaneous up- and down-regulation of desired genes in non-model plants. This method provides a powerful tool for functional genomics of medicinal plants, facilitating the discovery and production of valuable therapeutic compounds.

Partial desensitization of MYC2 transcription factor alters the interaction with jasmonate signaling components and affects specialized metabolism.

The activity of bHLH transcription factor MYC2, a key regulator in jasmonate signaling and plant specialized metabolism, is sensitive to repression by JASMONATE-ZIM-domain (JAZ) proteins and co-activation by the mediator subunit MED25. The substitution of a conserved aspartic acid (D) to asparagine (N) in the JAZ-interacting domain (JID) of Arabidopsis MYC2 affects interaction with JAZ, although the mechanism remained unclear. The effects of the conserved residue MYC2D128 on interaction with MED25 have not been investigated. Using tobacco as a model, we generated all possible substitutions of aspartic acid 128 (D128) in NtMYC2a. NtMYC2aD128N partially desensitized the repression by JAZ proteins, while strongly interacting with MED25, resulting in increased expression of nicotine pathway genes and nicotine accumulation in tobacco hairy roots overexpressing NtMYC2aD128N compared to those overexpressing NtMYC2a. The proline substitution, NtMYC2aD128P, negatively affected transactivation and abolished the interaction with JAZ proteins and MED25. Structural modeling and simulation suggest that the overall stability of the JID binding pocket is a predominant cause for the observed effects of substitutions at D128. The D128N substitution has an overall stabilizing effect on the binding pocket, which is destabilized by D128P. Our study offers an innovative tool to increase the production of plant natural products.

Metabolic engineering to enhance the accumulation of bioactive flavonoids licochalcone A and echinatin in Glycyrrhiza inflata (Licorice) hairy roots.

Echinatin and licochalcone A (LCA) are valuable chalcones preferentially accumulated in roots and rhizomes of licorice (Glycyrrhiza inflata). The licorice chalcones (licochalcones) are valued for their anti-inflammatory, antimicrobial, and antioxidant properties and have been widely used in cosmetic, pharmaceutical, and food industries. However, echinatin and LCA are accumulated in low quantities, and the biosynthesis and regulation of licochalcones have not been fully elucidated. In this study, we explored the potential of a R2R3-MYB transcription factor (TF) AtMYB12, a known regulator of flavonoid biosynthesis in Arabidopsis, for metabolic engineering of the bioactive flavonoids in G. inflata hairy roots. Overexpression of AtMYB12 in the hairy roots greatly enhanced the production of total flavonoids (threefold), echinatin (twofold), and LCA (fivefold). RNA-seq analysis of AtMYB12-overexpressing hairy roots revealed that expression of phenylpropanoid/flavonoid pathway genes, such as phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and flavanone 3'-hydroxylase (F3'H), is significantly induced compared to the control. Transient promoter activity assay indicated that AtMYB12 activates the GiCHS1 promoter in plant cells, and mutation to the MYB-binding motif in the GiCHS1 promoter abolished activation. In addition, transcriptomic analysis revealed that AtMYB12 overexpression reprograms carbohydrate metabolism likely to increase carbon flux into flavonoid biosynthesis. Further, AtMYB12 activated the biotic defense pathways possibly by activating the salicylic acid and jasmonic acid signaling, as well as by upregulating WRKY TFs. The transcriptome of AtMYB12-overexpressing hairy roots serves as a valuable source in the identification of potential candidate genes involved in LCA biosynthesis. Taken together, our findings suggest that AtMYB12 is an effective gene for metabolic engineering of valuable bioactive flavonoids in plants.

Engineering Properties of Sweet Potato Starch for Industrial Applications by Biotechnological Techniques including Genome Editing.

Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes.

Reprogramming plant specialized metabolism by manipulating protein kinases.

Being sessile, plants have evolved sophisticated mechanisms to balance between growth and defense to survive in the harsh environment. The transition from growth to defense is commonly achieved by factors, such as protein kinases (PKs) and transcription factors, that initiate signal transduction and regulate specialized metabolism. Plants produce an array of lineage-specific specialized metabolites for chemical defense and stress tolerance. Some of these molecules are also used by humans as drugs. However, many of these defense-responsive metabolites are toxic to plant cells and inhibitory to growth and development. Plants have, thus, evolved complex regulatory networks to balance the accumulation of the toxic metabolites. Perception of external stimuli is a vital part of the regulatory network. Protein kinase-mediated signaling activates a series of defense responses by phosphorylating the target proteins and translating the stimulus into downstream cellular signaling. As biosynthesis of specialized metabolites is triggered when plants perceive stimuli, a possible connection between PKs and specialized metabolism is well recognized. However, the roles of PKs in plant specialized metabolism have not received much attention until recently. Here, we summarize the recent advances in understanding PKs in plant specialized metabolism. We aim to highlight how the stimulatory signals are transduced, leading to the biosynthesis of corresponding metabolites. We discuss the post-translational regulation of specialized metabolism and provide insights into the mechanisms by which plants respond to the external signals. In addition, we propose possible strategies to increase the production of plant specialized metabolites in biotechnological applications using PKs.