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Molecular dissection of factors controlling flux through pathways

Final Report Summary - REGULATION OF FLUX (Molecular dissection of factors controlling flux through pathways)

Project context and objectives

The overall goal of the project was to understand the underlying molecular mechanisms controlling flux through biosynthetic pathways by using the glucosinolate pathway as a model system. In addition to three R2R3 MYB transcription factors (MYB28, MYB29 and MYB76), a late biosynthetic gene in the pathway, AOP2, had been suggested to control the levels of methionine-derived glucosinolates (GLS) and transcript the levels of many GLS biosynthetic genes as well as MYB28 and MYB29.

Work performed

To investigate whether the regulatory function of AOP2 is linked to the AOP2 transcript, the AOP2 protein or its enzymatic activity, different over-expression constructs were generated and introduced to Arabidopsis plants (accession Columbia 0) that lacked a functional AOP2. For one of the constructs, site-directed mutagenesis was used to ensure that no peptide could be produced from this transcript. Transgenic lines carrying this construct displayed on average twice the level of methionine-derived GLS than wild-type plants, thus indicating the presence of a long non-coding RNA with the capacity to regulate the pathway in the absence of the AOP2 protein.

Over-expression of an enzymatically inactive AOP2 protein was achieved by eliminating the iron-binding amino acid residues in the active site of the enzyme. The respective transgenic lines (accumulating the transcript and the inactive protein) did not hint at an additional regulatory role of the AOP2 protein. Finally, the plants that were transformed with a construct carrying the wild-type AOP2 gene expressed an enzymatically functional AOP2, which converted two short-chained methylsulfinylalkyl-GLS (3msp, 3-methylsulfinylpropyl-GLS; 4msb, 4-methylsulfinylbutyl-GLS) into alkenyl GLS (2 propenyl- and 3 butenyl-GLS, respectively). The total glucosinolate levels in these plants were up to 20 times greater than the wildtype. The regulatory function of AOP2 can therefore to a large extent be attributed to its enzymatic function and the resulting changes in the GLS profile.

Candidate metabolites, which could be sensed by the plant and thereby exhibit a feedback role in the regulation of the flux through the GLS pathway, included AOP2 substrates (3msp and 4msb) and products (2 propenyl- and 3 butenyl GLS). In the Arabidopsis Col 0 background, 3 butenyl GLS is subsequently oxidised by GLS OH to 2 hydroxy-3-butenyl-GLS. A possible role of the latter compound in GLS regulation could be ruled out based on the observation that over-expression of an enzymatically functional AOP2 in Col 0 wild-type plants revealed undistinguishable GLS levels.

To study the interplay between AOP2 and MYB28/MYB29/MYB76 in the regulatory network controlling glucosinolate biosynthesis, the capacity of AOP2 in various mutants and transgenic lines with altered MYB expression levels was investigated. The leaves of double knock-out (k.o.) plants lacking MYB28 and MYB29 accumulated only trace amounts of glucosinolates. Over-expression of AOP2 in this double k.o. did not result in an accumulation of glucosinolates. Thus, in contrast to the MYBs, AOP2 does not directly activate gene coding for enzymes in glucosinolate biosynthesis. Instead, AOP2 depends on the presence of MYB28 or MYB29 to regulate glucosinolate levels. MYB76 appears not to be critical for the regulatory function of AOP2. In the MYB28 single k.o. which accumulates lower levels of short-chained and long-chained GLS, over-expression of AOP2 resulted in increased levels of all GLS. Yet, AOP2 compensated only partially for the absence of MYB28 because wild-type levels were not reached. In the absence of MYB29, only the levels of short-chained GLS are lower than in wild-type leaves. Interestingly, AOP2 failed to increase biosynthesis of long-chained GLS level in the MTB29 k.o. background. Taken together, these findings shed light on the individual in planta roles of AOP2, MYB28 and MYB29 and thus allow for an extended model of glucosinolate regulation.