In vivo assembly of the sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana

Abstract

Summary Sorgoleone, a hydrophobic compound exuded from root hair cells of Sorghum spp., accounts for much of the allelopathic activity of the genus. The enzymes involved in the biosynthesis of this compound have been identified and functionally characterized. Here, we report the successful assembly of the biosynthetic pathway and the significant impact of in vivo synthesized sorgoleone on the heterologous host Nicotiana benthamiana. A multigene DNA construct was prepared for the expression of genes required for sorgoleone biosynthesis in planta and deployed in N. benthamiana leaf tissues via Agrobacterium‐mediated transient expression. RNA‐sequencing was conducted to investigate the effects of sorgoleone, via expression of its biosynthesis pathway, on host gene expression. The production of sorgoleone in agroinfiltrated leaves as detected by gas chromatography/mass spectrometry (GC/MS) resulted in the formation of necrotic lesions, indicating that the compound caused severe phytotoxicity to these tissues. RNA‐sequencing profiling revealed significant changes in gene expression in the leaf tissues expressing the pathway during the formation of sorgoleone‐induced necrotic lesions. Transcriptome analysis suggested that the compound produced in vivo impaired the photosynthetic system as a result of downregulated gene expression for the photosynthesis apparatus and elevated expression of proteasomal genes which may play a major role in the phytotoxicity of sorgoleone.

In planta Female Flower Agroinfiltration Alters the Cannabinoid Composition in Industrial Hemp (Cannabis sativa L.)

“…To date, agroinfiltration has been proven to be effective for metabolic engineering of representative secondary metabolites such as terpenoid (Reed and Osbourn, 2018 ; Mani et al, 2021 ), alkaloid (Li et al, 2019 ; Mora-Vasquez et al, 2022 ) carotenoid (Rodriguez-Concepcion and Daros, 2022 ), anthocyanin (Fresquet-Corrales et al, 2017 ) as well as minor secondary metabolites (van Herpen et al, 2010 ; Pan et al, 2021 ), most of which were achieved in N. benthamiana. This plant species shares typical cellular compartmentalization, cofactor, and coenzymes with other plants, enabling it to introduce new synthetic pathways without extensively optimizing the system.…”

Getting back to the grass roots: harnessing specialized metabolites for improved crop stress resilience

“…Alkylresorcinols are antimicrobial, allelochemicals suppressing neighboring plants, drivers of mycorrhizal colonization and mediators of rhizosphere compositions of nitrifying microorganisms [51,52]. Consistent with phytotoxic action, sorgoleone pathway engineering in tobacco (Nicotiana benthamiana) using a fatty acid desaturase, alkylresorcinol synthase family type III PKS, O-methyltransferase and SbCYP71AM1 resulted in necrosis and cell death [16].…”

“…Lipophilic GRSM are not commonly associated with glandular trichomes or secretory cavities, as is common in dicots, and thus are likely to be associated with either lipid droplets present in all forms of life or secreted into the soil [13]. Some GRSM are highly tissue-specific [14][15][16]; however, GRSM can also commonly co-occur in foliar tissues. Predictably, GRSM display pangenome variation in biochemical diversity consistent with complex selection pressures [11,12].…”

“…10-OPEA is broadly toxic to insects, CoA, the alkylresorcinol termed sorgoleone is a dominant sorghum root metabolite(Fig. 1) [16]. Diverse alkylresorcinols further occur in wheat, rye, barley and rice.…”

Variation on a theme: the structures and biosynthesis of specialized fatty acid natural products in plants

“…In the case of the sorgoleone pathway, the substrate used by the type III PKS is an unsaturated fatty acid (C16:3) generated from palmitic acid by two desaturases (SbDES2 and SbDES3; Pan et al., 2007). Subsequent action by an O ‐methyltransferase (Baerson et al., 2008) and a P450 (Pan et al., 2018) complete the pathway to sorgoleone, and all these enzymes together can be used to produce the compound heterologously (Pan et al., 2021), opening an avenue to engineering root–microbe interactions. Like those in Sorghum , PKS enzymes in Arabidopsis have been shown to act on long‐chain fatty acid substrates, though they use a slightly different cyclization mechanism and thereby generate tri‐ and tetraketide pyrones (AtPKS; Figure 3h; Dobritsa et al., 2010; Kim et al., 2010).…”