Structural Studies of Aliphatic Glucosinolate Chain-Elongation Enzymes

Plants evolved specialized metabolic pathways through gene duplication and functional divergence of enzymes involved in primary metabolism. The results of this process are varied pathways that produce an array of natural products useful to both plants and humans. In plants, glucosinolates are a diverse class of natural products. Glucosinolate function stems from their hydrolysis products, which are responsible for the strong flavors of Brassicales plants, such as mustard, and serve as plant defense molecules by repelling insects, fighting fungal infections, and discouraging herbivory. Additionally, certain hydrolysis products such as isothiocyanates can potentially serve as cancer prevention agents in humans. The breadth of glucosinolate function is a result of its great structural diversity, which comes from the use of aliphatic, aromatic and indole amino acids as precursors and elongation of some side chains by up to nine carbons, which, after the formation of the core glucosinolate structure, can undergo further chemical modifications. Aliphatic methionine-derived glucosinolates are the most abundant form of these compounds. Although both elongation and chemical modification of amino acid side chains are important for aliphatic glucosinolate diversity, its elongation process has not been well described at the molecular level. Here, we summarize new insights on the iterative chain-elongation enzymes methylthioalkylmalate synthase (MAMS) and isopropylmalate dehydrogenase (IPMDH).

Induction of Glucoraphasatin Biosynthesis Genes by MYB29 in Radish (Raphanus sativus L.) Roots

Glucoraphasatin (GRH) is a specific aliphatic glucosinolate (GSL) that is only abundant in radish (Raphanus sativus L.). The gene expression regulating GRH biosynthesis in radish is still poorly understood. We employed a total of 59 radish accessions to analyze GSL profiles and showed that GRH was specific and predominant among the aliphatic GSLs in radish roots. We selected five accessions roots with high, moderate and low GSL biosynthesis, respectively, to conduct a comparative transcriptome analysis and the qRT-PCR of the biosynthesis genes for aliphatic GSLs. In this study, among all the accessions tested, roots with the accession RA157-74 had a high GRH content and showed a significant expression of the aliphatic GSL biosynthesis genes. We defined the genes involved in the GRH biosynthesis process and found that they were regulated by a transcription factor (RSG00789) at the MYB29 locus in radish roots. We found 13 aliphatic GSL biosynthesis genes regulated by the RSG00789 gene in the GRH biosynthesis pathway.

CRISPR-Cas9-mediated editing of myb28 impairs glucoraphanin accumulation of Brassica oleracea in the field

SummaryWe sought to quantify the role of MYB28 in the regulation of aliphatic glucosinolate biosynthesis and associated sulphur metabolism in field-grown B. oleracea with the use of CRISPR-Cas9-mediated gene editing technology. We describe the first characterised myb28 knockout mutant in B. oleracea, and the first UK field trial of CRISPR-Cas9-mediated gene edited plants under the European Court of Justice interpretation of the 2001/18 EU GMO directive. We report that knocking-out myb28 results in downregulation of aliphatic glucosinolate biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants. There were no significant changes to the accumulation of sulphate, S-methyl cysteine sulfoxide and indole glucosinolate in leaf and floret tissues.

Epistatic Transcription Factor Networks Differentially Modulate Arabidopsis Growth and Defense

Plants integrate internal and external signals to finely coordinate growth and defense allowing for maximal fitness within a complex environment. One common model for the relationship between growth and defense is a trade-off model in which there is a simple negative interaction between growth and defense theoretically driven by energy costs. However, there is a developing consensus that the coordination of growth and defense likely involves a more conditional and intricate connection. To explore how a transcription factor network may coordinate growth and defense, we used high-throughput phenotyping to measure growth and flowering in a set of single and pairwise mutants previously linked to the aliphatic glucosinolate defense pathway. Showing the link between growth and aliphatic glucosinolate defense, 17 of the 20 tested TFs significantly influence plant growth and/or flowering time. These effects were conditional upon the environment, age of the plant and more critically varied amongst the phenotypes when using the same genotype. The phenotypic effects of the TF mutants on SC GLS accumulation and on growth did not display a simple correlation, supporting the coordination model. We propose that large transcription factor networks create a system to integrate internal and external signals and separately modulate growth and the accumulation of the defensive aliphatic GLS.