Metabolic effects of agro-infiltration on N. benthamiana accessions

Abstract

Over the recent years, Nicotiana benthamiana has gained great importance as a chassis for the production of high value, low volume pharmaceuticals and/or active pharmaceutical ingredients (APIs). The process involving infiltration of the N. benthamiana leaves with Agrobacterium spp, harbouring vectors with the gene of interest, facilitates transient expression. To date, little information is available on the effect of the agro-infiltration process on the metabolome of N. benthamiana, which is necessary to improve the process for large-scale, renewable manufacturing of high value compounds and medical products. Hence, the objective of the present study was to assess metabolic adaptation of N. benthamiana as a response to the presence of Agrobacterium. The present study elucidated changes of the steady-state metabolism in the agroinfiltrated leaf area, the area around the infection and the rest of the plant. Furthermore, the study discusses the phenotypic advantages of the N. benthamiana lab strain, optimised for agro-infiltration, compared to three other wild accessions. Results showed that the lab strain has a different metabolic composition and showed less alterations of the phenylpropanoid pathway and cell wall remodelling in the agroinfiltrated leaf areas, for example chlorogenic acid, cadaverine and C18:0–2-glycerol ester. In conclusion, both of these alterations present potential candidates to improve the phenotype of the N. benthamiana lab strain for a more efficient transient expression process.

The chemotype core collection of genus Nicotiana

“…All metabolites detected in the present study, except for unidentified molecular features, have been previously reported for individual Nicotiana species, mainly N. tabacum (Jassbi et al, 2017;Kaminski et al, 2020a). As previously reported for several N. benthamiana accessions, the present metabolite profiling did not detect any catechins or related/derived metabolites (Drapal et al, 2021b). This supports the hypothesis that the genus Nicotiana does not contain tannin synthesis, one of the major branches of the phenylpropanoid-derived biosynthesis (Vogt, 2010).…”

“…The extracts for three different analytical platforms were extracted with one methanol/water/chloroform separation method as previously described (Drapal et al., 2020b). The polar phase was aliquoted for metabolite profiling (100 μl) by UPLC‐ESI‐QToF 6560 (Agilent Technologies, Stockport, UK) and for metabolite profiling (150 μl) by GC‐MS detection (GC‐MSD) in splitless mode (Agilent Technologies) as previously described (Drapal et al., 2021b; Price et al., 2016). The extracts were spiked with internal standards genistein (1 μg) and d4‐succinic acid (10 μg).…”

A multi-omicNicotiana benthamianaresource for fundamental research and biotechnology

“…The QLD wild accession exhibits many morphological, developmental and metabolic differences to LAB 7,[14][15][16] , such as outcrossing flowers 7,14-16 , moth-attracting volatile production at night, and the robust capacity to produce anthocyanins (Figures 1C-D, S1, S2, Table S1). The levels of a range of metabolites such as phenolic acids, flavonoids, amino acid derivatives, and metabolites involved in defense responses [17][18][19][20] , such as nornicotine and hydroxygeranyl-linalool diterpene glycosides (HGL-DTG), exhibit marked differences between LAB and QLD (Figures 1E-F, S3, Table S2).…”

“…All of this work has relied on plants derived from one accession line (that we term LAB), that appears to have originated from a single collection near the Granites gold mine in central Australia 7,14,15 (Figure 1B). Several additional ecotypes have recently been described 7,14–16 .…”

“…All of this work has relied on plants derived from one accession line (that we term LAB), that appears to have originated from a single collection near the Granites gold mine in central Australia 7,14,15 (Figure 1B). Several additional ecotypes have recently been described 7,[14][15][16] .In this paper, we report chromosome level assemblies, epigenomic and metabolomic information for the LAB strain and the distantly related QLD ecotype, as well as their single nucleotide polymorphisms (SNPs) with two additional LAB and four additional wild accessions, providing a reference for the genus Nicotiana, and details about the genetic diversity of N. benthamiana. Furthermore, we study the genomic basis for their morphological, metabolic, and reproductive diversity, providing a comprehensive resource for fundamental research and biotechnology.…”