Identification and Characterization of the Role of Acetolactate Synthase 1 and Acylsugar Acyltransferases 1 and 2 in Acylsugar Biosynthesis in Nicotiana tabacum

Tobacco is one of the important commercial crops in the world, and it is also considered an efficient green bioreactor because of its amenability to transient and stable genetic transformations, as well as its high leaf biomass. In tobacco, acylsugars are specifically synthesized in glandular trichomes and are mainly tetra-acylsucroses, which represent the second major class of surface chemicals and their structures often differ from those in other species of the Solanaceae family. Tobacco acylsugars have been shown to have antioviposition, antifeedant, antimicrobial, antitumor, and other toxic properties which make them a wide range of potential commercial applications in pesticides, food and cosmetic additives etc. In addition, they are important precursors of flavor compounds and their degradation products contribute to the aroma quality of tobacco leaves. Therefore, the exploration of key genes of tobacco acylsugar biosynthesis and the analysis of their functional mechanisms are not only of great significance for tobacco resistance and quality breeding, but also can lay a certain foundation for the development and utilization of tobacco acylsugars. However, although great progress has been made in mining key genes involved in acylsugar biosynthesis in Solanum, Petunia and Salpiglossis, the genes and biosynthesis mechanisms of acylsugars have remained elusive in tobacco.
Previous studies in Solanaceae species have shown that the branched chain amino acid (BCAA) metabolic pathway is the main source of acylsugar acyl chains, and enzymes belonging to the acyltransferase family (BAHD) catalyze the attachment of acyl chains to sugar cores to form different types of acylsugars. Based on the research progress of acylsugar biosynthesis, through gene homology comparison and transcriptional expression level detection, our research group previously obtained three genes with high expression in glandular trichomes from tobacco, including one acetolactate synthase gene (NtALS1, one of the key gene involved in BCAA metabolic pathway) and two acylsugar acyltransferase gene (NtASAT1 and NtASAT2, belonging to BAHD family). In this thesis study, our research objective is to identify and characterize if these three genes are involved in the acylsugar biosynthesis of N. tabacum. The main results are as follows:
For NtALS1, tissue distribution analyses suggested that NtALS1 was highly expressed in the tips of glandular trichomes. Subcellular localization analysis showed that the NtALS1 localized to the chloroplast. Moreover, in the wild-type K326 variety background, we generated two NtALS1 loss-of-function mutants using the CRISPR-Cas9 system. Acylsugars contents in the two NtALS1 mutants were signifcantly lower than those in the wild type. Through phylogenetic tree analysis, we found that each acylsugar-containing Solanaceae species possessed an ALS homolog that clustered together with NtALS1, whereas the ALS homolog of N. sylvestris (one ancestor of N. tabacum), a species that does not contain acylsugars, did not cluster with NtALS1. Taken together, these findings indicate a functional role for NtALS1 in acylsugar biosynthesis in tobacco and also indicate that NtALS1 is likely from the ancestor N. tomentosiformis.
For NtASAT1 and NtASAT2, Tissue and subcellular localization analyses showed that both genes were trichome-specific and functioned in the endoplasmic reticulum. In vitro, NtASAT1 could utilize sucrose and C2-, iC4-, and iC5-CoA substrates to produce monoacylsucroses S1:2 (acetyl-CoA), S1:4 (iC4-CoA) and S1:5 (iC5-CoA), respectively, with the highest activity when iC5 was used as the acyl-donor. NMR analysis of the purified mono-acylsucrose (S1:5) revealed that the acyl group attached to position R2 of sucrose. NtASAT2 catalyzes the attachment of iC5 acyl-donor to iC5 mono-acylsucrose producing bi-acylsucrose (S2:10). These results were consistent with the fact that iC5 is more frequently found as an acyl side-chain in cultivated tobacco. Knocking-out of NtASAT1 or NtASAT2 led to the deficiency of acylsucrose in tobacco, however, no effect on the acylsugar accumulation was observed in NtASAT1 or NtASAT2 overexpression plants. Genomic and acylsugar profile analysis revealed that NtASAT1 and NtASAT2 are likely originated from diploid ancestor N. tomentosiformis producing acylsugars. These results revealed that NtASAT1 and NtASAT2 are key enzymes involved in the acylsugar assembly in tobacco.
This work extends our understanding of acylsucrose biosynthesis in tobacco plants, and also provides new genes for further study on molecular regulatory mechanisms of acylsugar biosynthesis, for the molecular breeding of tobacco aroma quality and engineering of pathogen- and insect-resistant crops.